Regulated transcription of targeted genes and other biological events

ABSTRACT

Dimerization and oligomerization of proteins are general biological control mechanisms that contribute to the activation of cell membrane receptors, transcription factors, vesicle fusion proteins, and other classes of intra- and extracellular proteins. We have developed a general procedure for the regulated (inducible) dimerization or oligomerization of intracellular proteins. In principle, any two target proteins can be induced to associate by treating the cells or organisms that harbor them with cell permeable, synthetic ligands. To illustrate the practice of this invention, we have induced: (1) the intracellular aggregation of the cytoplasmic tail of the ζ chain of the T cell receptor (TCR)-CD3 complex thereby leading to signaling and transcription of a reporter gene, (2) the homodimerization of the cytoplasmic tail of the Fas receptor thereby leading to cell-specific apoptosis (programmed cell death) and (3) the heterodimerization of a DNA-binding domain (Gal4) and a transcription-activation domain (VP16) thereby leading to direct transcription of a reporter gene. Regulated intracellular protein association with our cell permeable, synthetic ligands offers new capabilities in biological research and medicine, in particular, in gene therapy.

STATEMENT OF RIGHTS

This invention was made in the course of work supported by the U.S.Government. The U.S. Government therefore has certain rights in theinvention.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 08/196,043,filed Feb. 11, 1994, which is in turn a continuation-in-part of U.S.Ser. No. 08/179,748, filed Jan. 7, 1994, abandoned, which is in turn acontinuation-in-part of U.S. Ser. No. 08/092,977, filed Jul. 16, 1993,abandoned, which is in turn a continuation-in-part of U.S. Ser. No.08/017,931, filed Feb. 12, 1993, abandoned, and is in turn acontinuation-in-part of U.S. Ser. No. 08/292,597, filed Aug. 18, 1994,now U.S. Pat. No. 5,834,266 which is in turn a continuation-in-part ofU.S. Ser. No. 08/179,748, filed Jan. 7, 1994, abandoned, which is inturn a continuation-in-part of U.S. Ser. No. 08/092,977, filed Jul. 16,1993, abandoned, which is in turn a continuation-in-part of U.S. Ser.No. 08/017,931, filed Feb. 12, 1993, abandoned.

TECHNICAL FIELD

This invention concerns materials, methods and applications relating tothe oligomerizing of chimeric proteins with a dimeric or multimeric,preferably non-peptidic, organic molecule. Aspects of the invention areexemplified by recombinant modifications of host cells and their use ingene therapy or other applications of inducible gene expression.

INTRODUCTION

Biological specificity usually results from highly specific interactionsamong proteins. This principle is exemplified by signal transduction,the process by which extracellular molecules influence intracellularevents. Many pathways originate with the binding of extracellularligands to cell surface receptors. In many cases receptor dimerizationleads to transphosphorylation and the recruitment of proteins thatcontinue the signaling cascade. The realization that membrane receptorscould be activated by homodimerization resulted from the observationthat receptors could be activated by antibodies that cross linked tworeceptors. Subsequently, many receptors were found to share thoseproperties. The extracellular and transmembrane regions of manyreceptors are believed to function by bringing the cytoplasmic domainsof the receptors in close proximity by a ligand-dependent dimerizationor oligomerization, while the cytoplasmic domains of the receptor conveyspecific signals to internal compartments of the cell.

Others have investigated ligand-receptor interactions in differentsystems. For example, Clark, et al., Science (1992) 258, 123 describecytoplasmic effectors of the B-cell antigen receptor complex. Durand, etal., Mol. Cell. Biol. (1988) 8, 1715, Verweij, et al., J. Biol. Chem.(1990) 265, 15788 and Shaw, et al., Science (1988) 241, 202 report thatthe NF-AT-directed transcription is rigorously under the control of theantigen receptor. Inhibition of NF-AT-directed transcription bycyclosporin A and FK506 is reported by Emmel, et al., Science (1989)246, 1617 and Flanagan, et al., Nature (1991) 352, 803. Durand, et al.,Mol. Cell. Biol. (1988) 8, 1715 and Mattila, et al., EMBO J. (1990) 9,4425 describe the NF-AT binding sites. References describing the ζ chaininclude Orloff, et al., Nature (1990) 347, 189-191; Kinet, et al., Cell(1989) 57, 351-354; Weissman, et al., Proc. Natl. Acad. Sci. USA (1988)85, 9709-9713 and Lanier, Nature (1989) 342, 803-805. A CD4immunoadhesin is described by Byrn, et al. Nature (1990) 344, 667-670. ACD8-ζ-fused protein is described by Irving, et al., Cell (1992) 64, 891.See also, Letourner and Klausner, Science (1992) 255, 79.

Illustrative articles describing transcriptional factor association withpromoter regions and the separate activation and DNA binding oftranscription factors include: Keegan et al., Nature (1986) 231, 699;Fields and Song, ibid (1989) 340, 245; Jones, Cell (1990) 61, 9; Lewin,Cell (1990) 61, 1161; Ptashne and Gann, Nature (1990) 346, 329; Adamsand Workman, Cell (1993) 72, 306.

Illustrative articles describing vesicle targeting and fusion include:Sollner et al. (1993) Nature 362, 318-324; and Bennett and Scheller(1993) Proc. Natl. Acad. Sci. USA 90, 2559-2563.

Illustrative articles describing regulated protein degradation include:Hochstrasser et al (1990) Cell 61, 697; Scheffner, M. et al (1993) Cell75, 495; Rogers et al (1986) Science 234, 364-368.

Illustrative publications providing additional information concerningsynthetic techniques and modifications relevant to FK506 and relatedcompounds include: GB 2 244 991 A; EP 0 455 427 A1; WO 91/17754; EP 0465 426 A1, U.S. Pat. No. 5,023,263 and WO 92/00278.

However, as will be clear from this disclosure, none of the foregoingauthors describe or suggest the present invention. Our invention, whichis disclosed in detail hereinafter, involves a generally applicablemethod and materials for utilizing protein homodimerization,heterodimerization and oligomerization in living cells. Chimericresponder proteins are intracellularly expressed as fusion proteins witha specific receptor domain. Treatment of the cells with a cell permeablemultivalent ligand reagent which binds to the receptor domain leads todimerization or oligomerization of the chimeras. In analogy to otherchimeric receptors (see e.g. Weiss, Cell (1993) 73, 209), the chimericproteins are designed such that oligomerization triggers the desiredsubsequent events, e.g. the propagation of an intracellular signal viasubsequent protein-protein interactions and thereby the activation of aspecific subset of transcription factors. The initiation oftranscription can be detected using a reporter gene assay. Using genetransfer techniques to introduce artificial receptors, one can turn onor off the signaling pathways that lead to the over expression oftherapeutic proteins by administering orally active "dimerizers" or"de-dimerizers", respectively. Since cells from different recipients canbe configured to have the pathway over express different therapeuticproteins for use in a variety of disorders, the dimerizers have thepotential to serve as "universal drugs". They can also be viewed as cellpermeable, organic replacements for therapeutic antisense agents or forproteins that would otherwise require intravenous injection orintracellular expression (e.g., the LDL receptor or the CFTR protein).

Intracellular crosslinking of chimeric proteins by synthetic ligands haspotential in basic investigation of a variety of cellular processes andin regulating the synthesis of proteins of therapeutic or agriculturalimportance. Furthermore, ligand mediated oligomerization now permitsregulated gene therapy. In so doing, it provides a fresh approach toincreasing the safety, expression level and overall efficacy obtainedwith gene therapy.

SUMMARY OF THE INVENTION

This invention provides novel chimeric (or "fused") proteins and smallorganic molecules capable of oligomerizing the chimeric proteins. Thechimeric proteins contain at least one ligand-binding (or "receptor")domain fused to an additional ("action") domain, as described in detailbelow. As will also be described, the chimeric proteins may containadditional domains as well. These chimeric proteins are recombinant inthe sense that the various domains are derived from different sources,and as such, are not found together in nature (i.e., are heterologous).

Genes, i.e., RNA or preferably DNA molecules referred to herein as"genetic" or "DNA" constructs) which encode the novel chimeric proteins,and optionally target genes, are provided for the genetic engineering ofhost cells. Also provided are methods and compositions for producing andusing such modified cells. The engineered cells of this inventioncontain at least one such chimeric protein or a first series of geneticconstructs encoding the chimeric protein(s).These constructs arerecombinant in the sense that the component portions, e.g. encoding aparticular domain or expression control sequence, are not found directlylinked to one another in nature (i.e., are heterologous).

One DNA construct of this invention encodes a chimeric proteincomprising (a) at least one receptor domain (capable of binding to aselected ligand) fused to (b) a heterologous additional ("action")protein domain. Significantly, the ligand is capable of binding to two(or more) receptor domains, i.e. to chimeric proteins containing suchreceptor domains, in either order or simultaneously, preferably with aKd value below about 10⁻⁶, more preferably below about 10⁻⁷, even morepreferably below about 10⁻⁸, and in some embodiments below about 10⁻⁹ M.The ligand preferably is a non-protein and has a molecular weight ofless than about 5 kDa. The receptor domains of the chimeric proteins sooligomerized may be the same or different. The chimeric proteins arecapable of initiating a biological process upon exposure to the ligand,i.e., upon oligomerization with each other. The encoded chimeric proteinmay further comprises an intracellular targeting domain capable ofdirecting the chimeric protein to a desired cellular compartment. Thetargeting domain can be a secretory leader sequence, a membrane spanningdomain, a membrane binding domain or a sequence directing the protein toassociate with vesicles or with the nucleus, for instance.

The action domains of the chimeric proteins may be selected from a broadvariety of protein domains capable of effecting a desired biologicalresult upon oligomerization of the chimeric protein(s). For instance,the action domain may comprise a protein domain such as a CD3 zetasubunit capable, upon exposure to the ligand and subsequentoligomerization, of initiating a detectable intracellular signal; aDNA-binding protein such as Gal 4; or a transcriptional activationdomain such as VP16. Numerous other examples are provided herein. Oneexample of a detectable intracellular signal is a signal activating thetranscription of a gene under the transcriptional control of atranscriptional control element (e.g. enhancer/promoter elements and thelike) which is responsive to the oligomerization.

As is discussed in greater detail later, in various embodiments of thisinvention the chimeric protein is capable of binding to an FK506-typeligand, a cyclosporin A-type ligand, tetracycline or a steroid ligand.Such binding leads to oligomerization of the chimeric protein with otherchimeric protein molecules which may be the same or different.

Optionally the cells further contain a second recombinant geneticconstruct, or second series of such construct(s), containing a targetgene under the transcriptional control of a transcriptional controlelement (e.g. promoter/enhancer) responsive to a signal triggered byligand-mediated oligomerization of the chimeric proteins, i.e. toexposure to the ligand. These constructs are recombinant in the sensethat the target gene is not naturally under the transcriptional controlof the responsive transcriptional control element.

In one aspect of the invention the DNA construct contains (a) atranscriptional control element responsive to the oligomerization of achimeric protein as described above, and (b) flanking DNA sequence froma target gene permitting the homologous recombination of thetranscriptional control element into a host cell in association with thetarget gene. In other embodiments the construct contains a desired geneand flanking DNA sequence from a target locus permitting the homologousrecombination of the target gene into the desired locus. The constructmay also contain the responsive transcriptional control element, or theresponsive element may be provided by the locus. The target gene mayencodes a surface membrane protein, a secreted protein, a cytoplasmicprotein or a ribozyme or an antisense sequence.

The constructs of this invention may also contain a selectable markerpermitting transfection of the constructs into host cells and selectionof transfectants containing the construct. This invention furtherencompasses DNA vectors containing such constructs, whether for episomaltransfection or for integration into the host cell chromosomes. Thevetor may be a viral vector, including for example an adeno-, adenoassociated- or retroviral vector.

This invention further encompasses a chimeric protein encoded by any ofour DNA constructs, as well as cells containing and/or expressing them,including procaryotic and eucaryotic cells and in particular, yeast,worm, insect, mouse or other rodent, and other mammalian cells,including human cells, of various types and lineages, whether frozen orin active growth, whether in culture or in a whole organism containingthem.

For example, in one aspect, this invention provides cells, preferablybut not necessarly mammalian, which contain a first DNA constructencoding a chimeric protein comprising (i) at least one receptor domaincapable of binding to a selected oligomerizing ligand of this inventionand (ii) another protein domain, heterologous with respect to thereceptor domain, but capable, upon oligomerization with one or moreother like domains, of triggering the activation of transcription of atarget gene under the transcriptional control of a transcriptionalcontrol element responsive to said oligomerization. The cells furthercontain a target gene under the expresssion control of a transcriptionalcontrol element responsive to said oligomerization ligand. Followingexposure to the selected ligand expresses the target gene.

In another aspect, the invention provides cells which contain a firstset of DNA constructs encoding a first chimeric protein containing aDNA-binding domain and at least one receptor domain capable of bindingto a first selected ligand moiety. The cell further a second chimericprotein containing a transcriptional activating domain and at least onereceptor domain capable of binding to a second selected ligand (whichmay be the same or different from the first selected ligand moiety). Thecell additionally contains a DNA construct encoding a target gene underthe transcriptional control of a heterologous transcriptional controlsequence which binds with the DNA-binding domain and is responsive tothe transcriptional activating domain such that the cell expresses thetarget gene following exposure to a substance containing the selectedligand moiety(ies).

Also provided are a DNA composition comprising a first DNA constructencoding a chimeric protein comprising at least one receptor domain,capable of binding to a selected ligand, fused to a heterologousadditional protein domain capable of initiating a biological processupon exposure to the oligomerizing ligand, i.e. upon oligomerization ofthe chimeric protein; and a second DNA construct encoding a target geneunder the transcriptional control of a transcription control elementresponsive to the oligomerization ligand.

Another exemplary DNA composition of this invention comprises a firstseries of DNA constructs encoding a first and second chimeric proteinand a second DNA construct encoding a target gene under thetranscriptional control of an transcription control element responsiveto the oligomerization of the chimeric protein molecules. The DNAconstruct encoding the first chimeric protein comprises (a) at least onefirst receptor domain, capable of binding to a selected first ligandmoiety, fused to (b) a heterologous additional protein domain capable ofinitiating a biological process upon exposure to the oligomerizationligand, i.e. upon oligomerization of the first chimeric protein to asecond chimeric protein molecule. The DNA construct encoding the secondchimeric protein comprises (i) at least one receptor domain, capable ofbinding to a selected second ligand moiety, fused to (ii) a heterologousadditional protein domain capable of initiating a biological processupon exposure to the oligomerization ligand, i.e., upon oligomerizationto the first chimeric protein. The first and second receptor moieties insuch cases may be the same or different and the first and secondselected ligand moieties may likewise be the same or different.

Our ligands are molecules capable of binding to two or more chimericprotein molecules of this invention to form an oligomer thereof, andhave the formula:

    linker--{rbm.sub.1, rbm.sub.2, . . . rbm.sub.n }

wherein n is an integer from 2 to about 5, rbm.sub.(1) -rbm.sub.(n) arereceptor binding moieties which may be the same or different and whichare capable of binding to the chimeric protein(s). The rbm moieties arecovalently attached to a linker moiety which is a bi- ormulti-functional molecule capable of being covalently linked ("--") totwo or more rbm moieties. Preferably the ligand has a molecular weightof less than about 5 kDa and is not a protein. Examples of such ligandsinclude those in which the rbm moieties are the same or different andcomprise an FK506-type moiety, a cyclosporin-type moiety, a steroid ortetracycline. Cyclosporin-type moieties include cyclosporin andderivatives thereof which are capable of binding to a cyclophilin,naturally occurring or modified, preferably with a Kd value below about10⁻⁶ M. In some embodiments it is preferred that the ligand bind to anaturally occurring receptor with a Kd value greater than about 10⁻⁶ Mand more preferably greater than about 10⁻⁵ M. Illustrative ligands ofthis invention are those in which at least one rbm comprises a moleculeof FK506, FK520, rapamycin or a derivative thereof modified at C9, C10or both, which ligands bind to a modified receptor or chimeric moleculecontaining a modified receptor domain with a Kd value at least one, andpreferably 2, and more preferably 3 and even more preferably 4 or 5 ormore orders of magnitude less than their Kd values with respect to anaturally occurring receptor protein. Linker moiteies are also describedin detail later, but for the sake of illustration, include such moietiesas a C2-C20 alkylene, C4-C18 azalkylene, C6-C24 N-alkylene azalkylene,C6-C18 arylene, C8-C24 ardialkylene or C8-C36 bis-carboxamido alkylenemoiety.

The monomeric rbm's of this invention, as well as compounds containingsole copies of an rbm, which are capable of binding to our chimericproteins but not effecting dimerization or higher order oligomerizationthereof (in view of the monomeric nature of the individual rbm) areoligomerization antagonists.

In one embodiment, genetically engineered cells of this invention can beused for regulated production of a desired protein. In that embodimentthe cells, engineered in accordance with this invention to express adesired gene under ligand-induced regulation, are grown in culture byconventional means. Addition of the ligand to the culture medium leadsto expression of the desired gene and production of the desired protein.Expression of the gene and production of the protein can then be turnedoff by adding to the medium an oligomerization antagonist reagent, as isdescribed in detail below. Alternatively, this invention can be used toengineer ligand-inducable cell death characteristics into cells. Suchengineered cells can then be eliminated from a cell culture after theyhave served their intended purposed (e.g. production of a desiredprotein or other product) by adding the ligand to the medium. Engineeredcells of this invention can also be used in vivo, to modify wholeorganisms, preferably animals, including humans, e.g. such that thecells produce a desired protein or other result within the animalcontaining such cells. Such uses include gene therapy. Alternatively,the chimeric proteins and oligomerizing molecules can be usedextracellularly to bring together proteins which act in concert toinitiate a physiological action.

This invention thus provides materials and methods for achieving abiological effect in cells in response to the addition of anoligomerizing ligand. The method involves providing cells engineered inaccordance with this invention and exposing the cells to the ligand.

For example, one embodiment of the invention is a method for activatingtranscription of a target gene in cells. The method involves providingcells containing and capable of expressing (a) at least one DNAconstruct encoding a chimeric protein of this invention and (b) a targetgene. The chimeric protein comprises at least one receptor domaincapable of binding to a selected oligomerization ligand. The receptordomain is fused to an action domain capable--upon exposure to theoligomerizing ligand, i.e., upon oligomerization with one or more otherchimeric proteins containing another copy of the action domain--ofinitiating an intracellular signal. That signal is capable of activatingtranscription of a gene, such as the target gene in this case, which isunder the transcriptional control of a transcriptional control elementresponsive to that signal. The method thus involves exposing the cellsto an oligomerization ligand capable of binding to the chimeric proteinin an amount effective to result in expression of the target gene. Incases in which the cells are growing in culture, exposing them to theligand is effected by adding the ligand to the culture medium. In casesin which the cells are present within a host organism, exposing them tothe ligand is effected by administering the ligand to the host organism.For instance, in cases in which the host organism is an animal, inparticular, a mammal (including a human), the ligand is administered tothe host animal by oral, bucal, sublingual, transdermal, subcutaneous,intramuscular, intravenous, intra-joint or inhalation administration inan appropriate vehicle therefor.

This invention further encompasses a pharmaceutical compositioncomprising an oligomerization ligand of this invention in admixture witha pharmaceutically acceptable carrier and optionally with one or morekpharmaceutically aceptable excipients for activating the transcriptionof a target gene, for example, or effecting another biolotical result ofthis invention, in a subject containing engineered cells of thisinvention. The oligomerization ligand can be a homo-oligomerizationreagent or a heteo-oligomerization reagent as described in detailelsewhere. Likewise, this invention further encompasses a pharmaceuticalcomposition comprising an oligomerization antagonist of this inventionadmixture with a pharmaceutically acceptable carrier and optionally withone or more pharmaceutically acceptable excipients for reducing, inwhole or part, the level of oligomerization of chimerica proteins inengineered cells of this invention in a subject, and thus forde-activating the transcription of a target gene, for example, orturning off another biological result of this invention. Thus, the useof the oligomerizatoin reagents and of the oligomerization antagonistreagents to prepare pharmaceutical compositions is encompassed by thisinvention.

This invention also offers a method for providing a host organism,preferably an animal, and in many cases a mammal, responsive to anoligomerization ligand of this invention. The method involvesintroducing into the organism cells which have been engineered inaccordance with this invention, i.e. containing a DNA construct encodinga chimeric protein hereof, and so forth. Alternatively, one canintroduce the DNA constructs of this invention into a host organism,e.g. mammal under conditions permitting transfection of one or morecells of the host mammal.

We further provide kits for producing cells responsive to a ligand ofthis invention. One kit contains at least one DNA construct encoding oneof our chimeric proteins containing at least one receptor domain and anaction domain (as they are described elsewhere). The kit may contain aquantity of a ligand of this invention capable of oligomerizing thechimeric protein molecules encoded by the DNA constructs of the kit, andmay contain in addition a quantity of an oligomerization antagonist,e.g. monomeric ligand reagent. Where a sole chimeric protein is encodedby the construct(s), the oligomerization ligand is ahomo-oligomerization ligand. Where more than one such chimeric proteinis encoded, a hetero-oligomerization ligand may be included. The kit mayfurther contain a "second series" DNA construct encoding a target geneand/or transcription control element responsive to oligomerization ofthe chimeric protein molecules. The DNA constructs will preferably beassociated with one or more selection markers for convenient selectionof transfectants, as well as other conventional vector elements usefulfor replication in prokaryotes, for expression in eukaryotes, and thelike. The selection markers may be the same or different for eachdifferent DNA construct, permitting the selection of cells which containeach such DNA construct(s).

For example, one kit of this invention contains a first DNA constructencoding a chimeric protein containing at least one receptor domain(capable of binding to a selected ligand), fused to a transcriptionalactivator domain; a second DNA construct encoding a second chimericprotein containing at least one receptor domain (capable of binding to aselected ligand), fused to a DNA binding domain; and a third DNAconstruct encoding a target gene under the control of a transcriptionalcontrol element containing a DNA sequence to which the DNA bindingdomain binds and which is transcriptionally activated by exposure to theligand in the presence of the first and second chimeric proteins.

Alternatively, a DNA construct for introducing a target gene under thecontrol of a responsive transcriptional control element may contain acloning site in place of a target gene to provide a kit for engineeringcells to inducably express a gene to be provided by the practitioner.

Other kits of this invention may contain one or two (or more) DNAconstructs for chimeric proteins in which one or more contain a cloningsite in place of an action domain (transcriptional initiation signalgenerator, transcriptional activator, DNA binding protein, etc.),permitting the user to insert whichever action domain she wishes. Such akit may optionally include other elements as described above, e.g. DNAconstruct for a target gene under responsive expression control,oligomerization ligand, antagonist, etc.

Any of the kits may also contain positive control cells which werestably transformed with constructs of this invention such that theyexpress a reporter gene (for CAT, beta-galactosidase or any convenientlydetectable gene product) in response to exposure of the cells to theligand. Reagents for detecting and/or quantifying the expression of thereporter gene may also be provided.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram of the plasmid pSXNeo/IL2 (IL2-SX). In NF-AT-SX, theHindIII-ClaI DNA fragment from IL2-SX containing the IL2enhancer/promoter, is replaced by a minimal IL-2 promoter conferringbasal transcription and an inducible element containing three tandemNFAT-binding sites (described below).

FIG. 2 is a flow diagram of the preparation of the intracellularsignaling chimera plasmids p#MXFn and p#MFnZ, where n indicates thenumber of binding domains.

FIGS. 3A and 3B are a flow diagram of the preparation of theextracellular signalling chimera plasmid p#1FK3/pBJ5.

FIGS. 4A, 4B and 4C are sequences of the primers used in theconstructions of the plasmids employed in the subject invention SEQ IDNOS:4-40!.

FIG. 5 is a chart of the response of reporter constructs havingdifferent enhancer groups to reaction of the receptor TAC/CD3 ζ with aligand.

FIG. 6A is a chart of the activity of various ligands with the TAgJurkat cells described in Example 1. FIG. 6B illustrates geneticconstructs encoding various chimeric proteins including ligand bindingdomains from FKBP12.

FIG. 7 is a chart of the activity of the ligand FK1012A (8, FIG. 9B)with the extracellular receptor 1FK3 (FKBPx3/CD3 ζ).

FIG. 8 is a chart of the activation of an NFAT reporter via signallingthrough a myristoylated CD3 ζ/FKBP12 chimera.

FIGS. 9A, 9B, 9C, and 9D are the chemical structures of the allyl-linkedFK506 variants and the cyclohexyl-linked FK506 variants, respectively.

FIG. 10 is a flow diagram of the synthesis of derivatives of FK520.

FIGS. 11A, 11B, and 11C; are a flow diagram of a synthesis ofderivatives of FK520 and chemical structures of FK520, where the bottomstructures are designed to bind to mutant FKBP12.

FIG. 12 is a diagrammatic depiction of mutant FKBP with a modified FK520in the putative cleft.

FIGS. 13A and 13B is a flow diagram of the synthesis of heterodimers ofFK520 and cyclosporin.

FIG. 14 is a schematic representation of the oligomerization of chimericproteins, illustrated by chimeric proteins containing an immunophilinmoiety as the receptor domain.

FIG. 15 depicts ligand-mediated oligomerization of chimeric proteins,schowing schematically the triggering of a transcriptional initiationsignal.

FIGS. 16A and 16B depict synthetic schemes for HED and HOD reagentsbased on FK-506-type moieties. Panel A: FK1012s. Panel B: FK506 Monomerwith C10 Bump. Panel C: FK506 Monomer with a C9 Bump. Panel D: HEDReagent Synthesis.

FIG. 17 depicts the synthesis of (CsA)2 beginning with CsA.

FIGS. 18A-18B are an overview of the fusion cDNA construct and protienMZF3E.

FIG. 19 depicts co-immunoprecipitation of MZF1E_(h) with MZF1E_(f) inthe presence of FK1012 (E_(h) : Flu-epitop-tag, E_(f) :Flag-epitop-tag).

FIG. 20 shows FK1012-induced cell death of the Jurkat T-cell linetransfected with a myristoylated Fas-FKBP12 fusion protein (MFF3E), asindicated by the decreased transcriptional activity of the cells.

FIG. 21A is an analysis of cyclophilin-Fas (and Fas-cyclophilin) fusionconstructs in the transient transfection assay. MC3FE was shown to bethe most effective in this series.

FIG. 21B depicts Immunphilin-Fas antigen chimeras and results oftransient expression experiments in Jurkat T cells stably transformedwith large T-antigen. Myr: the myristylation sequence taken frompp^(60c-src) encoding residues 1-14 (Wilson et al, Mol & Cell Biol 9 4(1989): 1536-44); FKBP: human FKBP12; CypC: murine cyclophilin Csequence encoding residues 36-212 (Freidman et al, Cell 66 4 (1991):799-806); Fas: intracellular domain of human Fas antigen encodingresidues 179-319 (Oehm et al, J Biol Chem 267 15 (1992): 10709-15).Cells were electroporated with a plasmid encoding a secreted alkalinephosphatase reporter gene under the control of 3 tandem AP1 promotersalong with a six fold molar excess of the immunophilin fusion construct.After 24 h the cells were stimulated with PMA (50 ng/mL), whichstimulates the synthesis of the reporter gene, and (CsA)2. At 48 h thecells were assayed for reporter gene activity. Western blots wereperformed at 24 h using anti-HA epitope antibodies.

FIG. 22 depicts CAT assay results from Example 8.

FIGS. 23A and 23B depicts the synthesis of modified FK-506 typecompounds.

DESCRIPTION

I. Generic Discussion

This invention provides chimeric proteins, organic molecules foroligomerizing the chimeric proteins and a system for using them. Thefused proteins have a binding domain for binding to the (preferablysmall) organic oligomerizing molecules and an action domain, which caneffectuate a physiological action or cellular process as a result ofoligomerization of the chimeric proteins.

The basic concept for inducible protein association is illustrated inFIG. 14. Ligands which can function as heterodimerization (orhetero-oligomerization, "HED") and homodimerization (orhomo-oligomerization, "HOD") agents are depicted as dumbell-shapedstructures.

(Homodimerization and homo-oligomerization refer to the association oflike components to form dimers or oligomers, linked as they are by theligands of this invention. Heterodimerization and hetero-oligomerizationrefer to the association of dissimilar components to form dimers oroligomers. Homo-oligomers thus comprise an association of multiplecopies of a particular component while hetero-oligomers comprise anassociation of copies of different components. "Oligomerization","oligomerize" and "oligomer", as the terms are used herein, with orwithout prefixes, are intended to encompass "dimerization", "dimerize"and "dimer", absent an explicit indication to the contrary.)

Also depicted in FIG. 14 are fusion protein molecules containing atarget protein domain of interest ("action domain") and one or morereceptor domains that can bind to the ligands. For intracellularchimeric proteins, i.e., proteins which are located within the cells inwhich they are produced, a cellular targeting sequence (includingorganelle targeting amino acid sequences) will preferably also bepresent. Binding of the ligand to the receptor domains hetero- orhomodimerizes the fusion proteins. Oligomerization brings the actiondomains into close proximity with one another thus triggering cellularprocesses normally associated with the respective action domain--such asTCR-mediated signal transduction, for example.

Cellular processes which can be triggered by oligomerization include achange in state, such as a physical state, e.g. conformational change,change in binding partner, cell death, initiation of transcription,channel opening, ion release, e.g. Ca⁺² etc. or a chemical state, suchas a chemical reaction, e.g. acylation, methylation, hydrolysis,phosphorylation or dephosphorylation, change in redox state,rearrangement, or the like. Thus, any such process which can betriggered by ligand-mediated oligomerization is included within thescope of this invention.

As a first application of the subject invention, cells are modified soas to be responsive to the oligomerizing molecules. The modified cellscan be used in gene therapy, as well as in other applications whereinducible transcription or translation (both are included under the termexpression) is desired. The cells are characterized by a genomecontaining at least a first or first series (the series may include onlyone construct) of genetic constructs, and desirably a second or secondseries (the series may include only one construct) of constructs.

The nature and number of such genetic constructs will depend on thenature of the chimeric protein and the role it plays in the cell. Forinstance, in embodiments where the chimeric protein is to be associatedwith expression of a gene (and which may contain an intracellulartargeting sequence or domain which directs the chimeric protein to beassociated with the cellular surface membrane or with an organelle e.g.nucleus or vesicle), then there will normally be at least two series ofconstructs: a first series encoding the chimeric protein(s) which uponligand-mediated oligomerization initiate a signal directing target geneexpression, and desirably a second series which comprise the target geneand/or expression control elements therefor which are responsive to thesignal.

Only a single construct in the first series will be required where ahomooligomer, usually a homodimer, is involved, while two or more,usually not more than three constructs may be involved, where aheterooligomer is involved. The chimeric proteins encoded by the firstseries of constructs will be associated with actuation of genetranscription and will normally be directed to the surface membrane orthe nucleus, where the oligomerized chimeric protein is able toinitiate, directly or indirectly, the transcription of one or moretarget genes. A second series of additional constructs will be requiredwhere an exogenous gene(s) is introduced, or where an exogenous orrecombinant expression control sequence is introduced (e.g. byhomologous recombination) for expression of an endogenous gene, ineither case, whose transcription will be activated by the oligomerizingof the chimeric protein.

A different first series of constructs are employed where the chimericproteins are intracellular and can act directly without initiation oftranscription of another gene. For example, proteins associated withexocytosis can be expressed inducibly or constitutively, where theproteins will not normally complex except in the presence of theoligomerizing molecule. By employing proteins which have any or all ofthese properties which do not complex in the host cell; are inhibited bycomplexation with other proteins, which inhibition may be overcome byoligomerization with the ligand; require activation through a processwhich is not available in the host cell; or by modifying the proteinswhich direct fusion of a vesicle with the plasma membrane to formchimeric proteins, where the extent of complex formation and membranefusion is enhanced in the presence of the oligomerizing molecule,exocytosis is or has the ability to be induced b the oligomerizingmolecule.

Other intracellular proteins, such as kinases, phosphatases and cellcycle control proteins can be similarly modified and used.

Various classes of genetic constructs of this invention are described asfollows:

(1) constructs which encode a chimeric protein comprising a bindingdomain and an action domain, where the binding domain is extracellularor intracellular and the action domain is intracellular such thatligand-mediated oligomerization of the chimeric protein, by itself (toform a homo-oligomer) or with a different fused protein comprising adifferent action domain (to form a hetero-oligomer), induces a signalwhich results in a series of events resulting in transcriptionalactivation of one or more genes;

(2) constructs which encode a chimeric protein having a binding domainand an action domain, where the binding domain and action domain are inthe nucleus, such that ligand-mediated oligomerization of the protein,by itself (to form a homo-oligomer) or with a different fused proteincomprising a different action domain (to form a hetero-oligomer),induces initiation of transcription directly via complexation of theoligomer(s) with the DNA transcriptional initiation region;

(3) constructs which encode a chimeric protein containing a bindingdomain and an action domain, where the binding domain and the actiondomain are cytoplasmic, such that ligand-mediated oligomerization of theprotein, by itself (to form a homo-oligomer) or with a different fusedprotein comprising a different action domain (to form ahetero-oligomer), results in exocytosis; and

(4) constructs which encode a chimeric protein containing a bindingdomain and an action domain, where the binding domain and action domainare extracellular and the action domain is associated with initiating abiological activity (by way of non-limiting illustration, the actiondomain can itself bind to a substance, receptor or other membraneprotein yielding, upon ligand-mediated oligomerization of the chimeras,the bridging of one or more similar or dissimilar molecules or cells);and,

(5) constructs which encode a destabilizing, inactivating or short-livedchimeric protein having a binding domain and an action domain, such thatligand-mediated oligomerization of the protein with a target proteincomprising a different action domain leads to the destabilization and/ordegradation or inactivation of said oligomerized target protein.

II. Transcription Regulation

The construct(s) of Groups (1) and (2), above, will be considered first.Group (1) constructs differ from group (2) constructs in their effect.Group (1) constructs are somewhat pleiotropic, i.e. capable ofactivating a number of wild-type genes, as well as the target gene(s).In addition, the response of the expression products of group (1) genesto the ligand is relatively slow. Group (2) constructs can be directedto a specific target gene and are capable of limiting the number ofgenes which will be transcribed. The response of expression products ofgroup (2) constructs to the ligand is very rapid.

The subject system for groups (1) and (2) will include a first series ofconstructs which comprise DNA sequences encoding the chimeric proteins,usually involving from one to three, usually one to two, differentconstructs. The system usually will also include a second series ofconstructs which will provide for expression of one or more genes,usually an exogenous gene. By "exogenous gene" is meant a gene which isnot otherwise normally expressed by the cell, e.g. because of the natureof the cell, because of a genetic defect of the cell, because the geneis from a different species or is a mutated or synthetic gene, or thelike. Such gene can encode a protein, antisense molecule, ribozyme etc.,or can be a DNA sequence comprising an expression control sequencelinked or to be linked to an endogenous gene with which the expressioncontrol sequence is not normally associated. Thus, as mentioned before,the construct can contain an exogenous or recombinant expression controlsequence for ligand-induced expression of an endogenous gene.

The chimeric protein encoded by a construct of groups (1), (2) and (3)can have, as is often preferred, an intracellular targeting domaincomprising a sequence which directs the chimeric protein to the desiredcompartment, e.g. surface membrane, nucleus, vesicular membrane, orother site, where a desired physiological activity can be initiated bythe ligand-mediated oligomerization, at least dimerization, of thechimeric protein.

The chimeric protein contains a second ("binding" or "receptor") domainwhich is capable of binding to at least one ligand molecule. Since theligand can contain more than one binding site or epitope, it can formdimers or higher order homo- or hetero-oligomers with the chimericproteins of this invention. The binding domain of the chimeric proteincan have one or a plurality of binding sites, so that homooligomers canbe formed with a divalent ligand. In this way the ligand can oligomerizethe chimeric protein by having two or more epitopes to which the seconddomain can bind, thus providing for higher order oligomerization of thechimeric protein.

The chimeric protein also contains a third ("action") domain capable ofinitiating a biological activity upon ligand-mediated oligomerization ofchimeric protein molecules via the binding domains. Thus, the actiondomain may be associated with transduction of a signal as a result ofthe ligand-mediated oligomerization. Such signal, for instance, couldresult in the initiation of transcription of one or more genes,depending on the particular intermediate components involved in thesignal transduction. See FIG. 15 which depicts an illustrative chimericprotein in which the intracellular targeting domain comprises amyristate moiety; the receptor domain comprises three FKBP12 moieties;and the action domain comprises a zeta subunit. In other chimericproteins the action domains may comprise transcription factors, whichupon oligomerization, result in the initiation of transcription of oneor more target genes, endogenous and/or exogenous. The action domainscan comprise proteins or portions thereof which are associated withfusion of vesicle membranes with the surface or other membrane, e.g.proteins of the SNAP and SNARE groups (See, Sollner et al. (1993) 362,318 and 353; Cell (1993) 72, 43).

A. Surface Membrane Receptor

Chimeric proteins of one aspect of this invention are involved with thesurface membrane and are capable of transducing a signal leading to thetranscription of one or more genes. The process involves a number ofauxiliary proteins in a series of interactions culminating in thebinding of transcription factors to promoter regions associated with thetarget gene(s). In cases in which the transcription factors bind topromoter regions associated with other genes, transcription is initiatedthere as well. A construct encoding a chimeric protein of thisembodiment can encode a signal sequence which can be subject toprocessing and therefore may not be present in the mature chimericprotein. The chimeric protein will in any event comprise (a) a bindingdomain capable of binding a pre-determined ligand, (b) an optional(although in many embodiments, preferred) membrane binding domain whichincludes a transmembrane domain or an attached lipid for translocatingthe fused protein to the cell surface/membrane and retaining the proteinbound to the cell surface membrane, and, (c) as the action domain, acytoplasmic signal initiation domain. The cytoplasmic signal initiationdomain is capable of initiating a signal which results in transcriptionof a gene having a recognition sequence for the initiated signal in thetranscriptional initiation region.

The gene whose expression is regulated by the signal from the chimericprotein is referred to herein as the "target" gene, whether it is anexogenous gene or an endogenous gene under the expression control of anendogenous or exogenous (or hybrid) expression control sequence. Themolecular portion of the chimeric protein which provides for binding toa membrane is also referred to as the "retention domain". Suitableretention domains include a moiety which binds directly to the lipidlayer of the membrane, such as through lipid participation in themembrane or extending through the membrane, or the like. In such casesthe protein becomes translocated to and bound to the membrane,particularly the cellular membrane, as depicted in FIG. 15.

B. Nuclear Transcription Factors

Another first construct encodes a chimeric protein containing a cellulartargeting sequence which provides for the protein to be translocated tothe nucleus. This ("signal consensus") sequence has a plurality of basicamino acids, referred to as a bipartite basic repeat (reviewed inGarcia-Bustos et al, Biochimica et Biophysica Acta (1991) 1071, 83-101).This sequence can appear in any portion of the molecule internal orproximal to the N- or C-terminus and results in the chimeric proteinbeing inside the nucleus. The practice of one embodiment of thisinvention will involve at least two ("first series") chimeric proteins:(1) one having an action domain which binds to the DNA of thetranscription initiation region associated with a target gene and (2) adifferent chimeric protein containing as an action domain, atranscriptional activation domain capable, in association with the DNAbinding domain of the first chimeric protein, of initiatingtranscription of a target gene. The two action domains or transcriptionfactors can be derived from the same or different protein molecules.

The transcription factors can be endogenous or exogenous to the cellularhost. If the transcription factors are exogenous, but functional withinthe host and can cooperate with the endogenous RNA polymerase (ratherthan requiring an exogenous RNA polymerase, for which a gene could beintroduced), then an exogenous promoter element functional with thefused transcription factors can be provided with a second construct forregulating transcription of the target gene. By this means theinitiation of transcription can be restricted to the gene(s) associatedwith the exogenous promoter region, i.e., the target gene(s).

A large number of transcription factors are known which require twosubunits for activity. Alternatively, in cases where a singletranscription factor can be divided into two separate functional domains(e.g. a transcriptional activator domain and a DNA-binding domain), sothat each domain is inactive by itself, but when brought together inclose proximity, transcriptional activity is restored. Transcriptionfactors which can be used include yeast GAL4, which can be divided intotwo domains as described by Fields and Song, supra. The authors use afusion of GAL4(1-147)-SNF1 and SNF4-GAL4(768-881), where the SNF1 and -4may be replaced by the subject binding proteins as binding domains.Combinations of GAL4 and VP16 or HNF-1 can be employed. Othertranscription factors are members of the Jun, Fos, and ATF/CREBfamilies, Oct1, Sp1, HNF-3, the steriod receptor superfamily, and thelike.

As an alternative to using the combination of a DNA binding domain and anaturally occurring activation domain or modified form thereof, theactivation domain may be replaced by one of the binding proteinsassociated with bridging between a transcriptional activation domain andan RNA polymerase, including but not limited to RNA polymerase II. Theseproteins include the proteins referred to as TAF's, the TFII proteins,particularly B and D, or the like. Thus, one can use any one orcombination of proteins, for example, fused proteins or binding motifsthereof, which serve in the bridge between the DNA binding protein andRNA polymerase and provide for initiation of transcription. Preferably,the protein closest to the RNA polymerase will be employed inconjunction with the DNA binding domain to provide for initiation oftranscription. If desired, the subject constructs can provide for threeor more, usually not more than about 4, proteins to be brought togetherto provide the transcription initiation complex.

Rather than have a transcriptional activation domain as an actiondomain, an inactivation domain, such as ssn-6/TUP-1 or Kruppel-familysuppressor domain, can be employed. In this manner, regulation resultsin turning off the transcription of a gene which is constitutivelyexpressed. For example, in the case of gene therapy one can provide forconstitutive expression of a hormone, such as growth hormone, bloodproteins, immunoglobulins, etc. By employing constructs encoding onechimeric protein containing a DNA binding domain joined to a ligandbinding domain and another chimeric protein containing an inactivationdomain joined to a ligand binding domain, the expression of the gene canbe inhibited via ligand-mediated oligomerization.

Constructs encoding a chimeric protein containing inter alia aligand-binding domain fused to a transcriptional activating domain orsubunit, transcriptional inactivating domain or DNA-binding domain aredesigned and assembled in the same manner as described for the otherconstructs. Frequently, the N-terminus of the transcription factor willbe bound to the C-terminus of the ligand-binding domain, although insome cases the reverse will be true, for example, where two individualdomains of a single transcription factor are divided between twodifferent chimeras.

III. Exocytosis

Another use for the ligand-mediated oligomerization mechanism isexocytosis, where export of a protein rather than transcription iscontrolled by the ligand. This can be used in conjunction with theexpression of one or more proteins of interest, as an alternative toproviding for secretion of the protein(s) of interest via a secretorysignal sequence. This embodiment involves two different firstconstructs. One construct encodes a chimeric protein which directs theprotein to the vesicle to be integrated into the vesicular membrane asdescribed by Sollner et al., supra. Proteins which may be used as thevesicle binding protein include VAMP (synaptobrevin), SNC2, rab3, SEC4,synaptotagmin, etc., individually or in combination. The cellularmembrane protein may include syntaxin, SSO1, SSO2, neurexin, etc.,individually or in combination. The other construct provides fortransport to the surface membrane and employs the myristoyl signalsequence, other plasma membrane targeting sequence (e.g. forprenylation) or transmembrane retention domain, as described above. Theencoded proteins are described in the above references and, all orfunctional part, may serve as the action domains. These constructs couldbe used in conjunction with the expression of an exogenous protein,properly encoded for transport to a vesicle or for an endocytoticendogenous protein, to enhance export of the endogenous protein.

Various mechanisms can be employed for exocytosis. Depending on the celltype and which protein is limiting for endocytosis in the cell, one ormore of the vesicle bound proteins or cellular proteins may be encodedby one or more constructs having a response element which is activatedby the ligand. Of particular interest is the combination of VAMP andsyntaxin. Alternatively, one can provide for constitutive expression ofnon-limiting proteins controlling exocytosis and provide for ligandregulated expression of the exocytosis limiting protein. Finally, onecan provide for constitutive expression of the chimeric proteinsassociated with exocytosis, so that exocytosis is controlled byoligomerizing the chimeric proteins with the ligand. By employingappropriate binding domains, one can provide for different chimericproteins to be oligomerized on the vesicle surface to form an activecomplex, and/or linking of the vesicle protein(s) with the cell membranesurface protein through the ligand. The chimeric proteins may notprovide for exocytosis in the absence of the ligand due to modificationsin the ligand which substantially reduce the binding affinity betweenthe proteins governing exocytosis, such as deletions, mutations, etc.These modifications can be readily determined by employing overlappingfragments of the individual proteins and determining which fragmentsretain activity. The fragments can be further modified by using alaninesubstitutions to determine the individual amino acids whichsubstantially affect binding. (Beohncke et al., J. Immunol. (1993) 150,331-341; Evavold et al., ibid (1992) 148, 347-353).

The proteins assembled in the lumen of the vesicle, as well as the fusedproteins associated with exocytosis can be expressed constitutively orinducibly, as described above. Depending on the purpose of theexocytosis, whether endogenous or exogenous proteins are involved,whether the proteins to be exported are expressed constitutively orindiucibly, whether the same ligand can be used for initiatingtranscription of the fused proteins associated with exocytosis and theproteins to be exported, or whether the different proteins are to besubject to different inducible signals, may determine the manner inwhich expression is controlled. In one aspect, the exocytosis mechanismwould be the only event controlled by the ligand. In other aspects, bothexpression of at least one protein and exocytosis may be subject toligand control.

Various proteins may be modified by introduction of a cellular targetingsequence for translocation of the protein to a vesicle without loss ofthe physiological activity of the protein. By using exocytosis as thedelivery mechanism, relatively high dosages may be delivered within ashort period of time to produce a high localized level of the protein ora high concentration in the vascular system, depending on the nature ofthe host. Proteins of interest include e.g. insulin, tissue plasminogenactivator, cytokines, erythropoietin, colony stimulating factors, growthfactors, inflammatory peptides, cell migration factors.

Coding sequences for directing proteins to a vesicle are available fromthe vesicle binding proteins associated with exocytosis. See, forexample, Sollner, et al. supra.

Another use of the oligomerization mechanism is the control of proteindegradation or inactivation. For example, a relatively stable orlong-lived chimeric protein of this invention can be destabilized ortargeted for degradation by ligand-mediated oligomerization with adifferent chimeric protein of this invention which has a relativelyshort half-life or which otherwise destabilizes or targets the oligomerfor degradation. In this embodiment, ligand-mediated oligomerizationregulates biological functioning of a protein by conferring upon it intrans a shortened half-life. The latter chimeric protein may contain adomain targeting the protein to the lysosome or a domain rendering theprotein susceptible to proteolytic cleavage in the cytosol or nucleus ornon-lysosomal organelle.

The half-life of proteins within cells is determined by a number offactors which include the presence of short amino acid sequences withinsaid protein rich in the amino acid residues proline, glutamic acid,serine and threonine, hence "PEST", other sequences with similarfunction, protease sensitive cleavage sites and the state ofubiquitinization. Ubiquitinization is the modification of a protein byone or more units of the short polypeptide chain, ubiquitin, whichtargets proteins for degradation. The rate of ubiquitinization ofproteins is considered to be determined primarily by the identity of theN-terminal amino acid of the processed protein and one or more uniquelysine residues near the amino terminus.

IV. Other Regulatory Systems

Other biological functions which can be controlled by oligomerization ofparticular activities associated with individual proteins are proteinkinase or phosphatase activity, reductase activity, cyclooxygenaseactivity, protease activity or any other enzymatic reaction dependent onsubunit association. Also, one may provide for association of G proteinswith a receptor protein associated with the cell cycle, e.g. cyclins andcdc kinases, multiunit detoxifying enzymes.

V. Components of Constructs

The second or additional constructs (target genes) associated with group(1) and (2) chimeric proteins comprise a transcriptional initiationregion having the indicated target recognition sequence or responsiveelement, so as to be responsive to signal initiation from the activatedreceptor or activated transcription factors resulting in at least onegene of interest being transcribed to a sequence(s) of interest, usuallymRNA, whose transcription and, as appropriate, translation may result inthe expression of a protein and/or the regulation of other genes, e.g.antisense, expression of transcriptional factors, expression of membranefusion proteins, etc.

For the different purposes and different sites, different bindingdomains and different cytoplasmic domains will be used. For chimericprotein receptors associated with the surface membrane, if theligand-binding domain is extracellular, the chimeric protein can bedesigned to contain an extracellular domain selected from a variety ofsurface membrane proteins. Similarly, different cytoplasmic orintracellular domains of the surface membrane proteins which are able totransduce a signal can be employed, depending on which endogenous genesare regulated by the cytoplasmic portion. Where the chimeric protein isinternal, internal to the surface membrane protein or associated with anorganelle, e.g. nucleus, vesicle, etc., the ligand-binding domainprotein will be restricted to domains which can bind molecules which cancross the surface membrane or other membrane, as appropriate. Therefore,these binding domains will generally bind to small naturally occurringor synthetic ligand molecules which do not involve proteins or nucleicacids.

A. Cytoplasmic domains

A chimeric protein receptor of Group (1) can contain a cytoplasmicdomain from one of the various cell surface membrane receptors,including muteins thereof, where the recognition sequence involved ininitiating transcription associated with the cytoplasmic domain is knownor a gene responsive to such sequence is known. Mutant receptors ofinterest will dissociate transcriptional activation of a target genefrom activation of genes which can be associated with harmful sideeffects, such as deregulated cell growth or inappropriate release ofcytokines. The receptor-associated cytoplasmic domains of particularinterest will have the following characteristics: receptor activationleads to initiation of transcription for relatively few (desirably fewerthan 100) and generally innocuous genes in the cellular host; the otherfactors necessary for transcription initated by receptor activation arepresent in the cellular host; genes which are activated other than thetarget genes will not affect the intended purpose for which these cellsare to be used; oligomerization of the cytoplasmic domain or otheravailable mechanism results in signal initiation; and joining of thecytoplasmic domain to a desired ligand-binding domain will not interferewith signalling. A number of different cytoplasmic domains are known.Many of these domains are tyrosine kinases or are complexed withtyrosine kinases, e.g. CD3 ζ, IL-2R, IL-3R, etc. For a review seeCantley, et al., Cell (1991) 64, 281. Tyrosine kinase receptors whichare activated by cross-linking, e.g. dimerization (based on nomenclaturefirst proposed by Yarden and Ulrich, Annu. Rev. Biochem. (1988) 57, 443,include subclass I: EGF-R, ATR2/neu, HER2/neu, HER3/c-erbB-3, Xmrk;subclass II: insulin-R, IGF-1-R insulin-like growth factor receptor!,IRR; subclass III: PDGF-R-A, PDGF-R-B, CSF-1-R (M-CSF/c-Fms), c-kit,STK-1/Flk-2; and subclass IV: FGF-R, flg acidic FGF!, bek basic FGF!);neurotrophic tryosine kinases: Trk family, includes NGF-R, Ror1,2.Receptors which associate with tyrosine kinases upon cross-linkinginclude the CD3 ζ-family: CD3 ζ and CD3 η (found primarily in T cells,associates with Fyn); β and γ chains of Fc.sub.ε RI (found primarily inmast cells and basophils); γ chain of Fc.sub.γ RIII/CD16 (foundprimarily in macrophages, neutrophils and natural killer cells); CD3 γ,-δ, and -ε (found primarily in T cells); Ig-α/MB-1 and Ig-β/B29 (foundprimarily in B cell). Many cytokine and growth factor receptorsassociate with common β subunits which interact with tyrosine kinasesand/or other signalling molecules and which can be used as cytoplasmicdomains in chimeric proteins of this invention. These include (1) thecommon β subunit shared by the GM-CSF, IL-3 and IL-5 receptors; (2) theβ chain gp130 associated with the IL-6, leukemia inhibitory factor(LIF), ciliary neurotrophic factor (CNTF), oncostatin M, and IL-11receptors; (3) the IL-2 receptor γ subunit associated also withreceptors for IL-4, 11, IL-7 and IL-13 (and possibly IL-9); and (4) theβ chain of the IL-2 receptor which is homologous to the cytoplasmicdomain of the G-CSF receptor.

The interferon family of receptors which include interferons α/β and γ(which can activate one or more members of the JAK, Tyk family oftyrosine kinases) as well as the receptors for growth hormone,erythropoietin and prolactin (which also can activate JAK2) can also beused as sources for cytoplasmic domains.

Other sources of cytoplasmic domains include the TGF-β family of cellsurface receptors (reviewed by Kingsley, D., Genes and Development 19948 133). This family of receptors contains serine/threonine kinaseactivity in their cytoplasmic domains, which are believed to be actiatedby crosslinking.

The tyrosine kinases associated with activation and inactivation oftranscription factors are of particular interest in providing specificpathways which can be controlled and can be used to initiate or inhibitexpression of an exogenous gene.

The following table provides a number of receptors and characteristicsassociated with the receptor and their nuclear response elements thatactivate genes. The list is not exhaustive, but provides exemplarysystems for use in the subject invention.

In many situations mutated cytoplasmic domains can be obtained where thesignal which is transduced may vary from the wild type, resulting in arestricted or different pathway as compared to the wild-type pathway(s).For example, in the case of growth factors, such as EGF and FGF,mutations have been reported where the signal is uncoupled from cellgrowth but is still maintained with c-fos (Peters, et al., Nature (1992)358, 678).

The tyrosine kinase receptors can be found on a wide variety of cellsthroughout the body. In contrast, the CD3 ζ-family, the Ig family andthe lymphokine β-chain receptor family are found primarily onhematopoietic cells, particularly T-cells, B-cells, mast cells,basophils, macrophages, neutrophils, and natural killer cells. Thesignals required for NF-AT transcription come primarily from the zeta(ζ) chain of the antigen receptor and to a lesser extent CD3γ, δ, ε.

                  TABLE 1    ______________________________________           DNA      Binding    Ligand Element  Factor(s)                             Gene   Reference    ______________________________________    Insulin           cAMP     LRFI     jun-B  Mol. Cell Biol. (1992),    and others           responsive        many   12, 4654           element           genes  PNAS, 83, 3439           (cre)    PDGF,  SRE      SRF/SR   c-fos  Mol. Cell Biol. (1992),    FGF, TGF        EBP             12, 4769    and others    EGF    VL30              RVL-3  Mol. Cell. Biol. (1992),           RSRF              virus  12, 2793                             c-jun  Mol. Cell. Biol. (1992),                                    12, 4472    IFN-α           ISRE     ISGF-3          Gene Dev. (1989) 3,                                    1362    IFN-γ           GAS      GAF      GBP    Mol. Cell. Biol. (1991)                                    11, 182    PMA and         AP-1     many   Cell (1987) 49, 729-739    TCR                      genes    TNF             NFκB                             many   Cell (1990) 62,                             genes  1019-1029    Antigen           ARRE-1   OAP/O    many   Mol. Cell. Biol. (1988)                    ct-1     genes  8, 1715    Antigen           ARRE-2   NFAT     IL-2   Science (1988) 241, 202                             enhancer    ______________________________________

The cytoplasmic domain, as it exists naturally or as it may betruncated, modified or mutated, will be at least about 10, usually atleast about 30 amino acids, more usually at least about 50 amino acids,and generally not more than about 400 amino acids, usually not more thanabout 200 amino acids. (See Romeo, et al., Cell (1992) 68, 889-893.)While any species can be employed, the species endogenous to the hostcell is usually preferred. However, in many cases, the cytoplasmicdomain from a different species can be used effectively. Any of theabove indicated cytoplasmic domains may be used, as well as others whichare presently known or may subsequently be discovered.

For the most part, the other chimeric proteins associated withtranscription factors, will differ primarily in having a cellulartargeting sequence which directs the chimeric protein to the internalside of the nuclear membrane and having transcription factors orportions thereof as the action domains. Usually, the transcriptionfactor action domains can be divided into "DNA binding domains" and"activation domains." One can provide for a DNA binding domain with oneor more ligand binding domains and an activation domain with one or moreligand binding domains. In this way the DNA binding domain can becoupled to a plurality of binding domains and/or activation domains.Otherwise, the discussion for the chimeric proteins associated with thesurface membrane for signal transduction is applicable to the chimericproteins for direct binding to genomic DNA. Similarly, the chimericprotein associated with exocytosis will differ primarily as to theproteins associated with fusion of the vesicle membrane with the surfacemembrane, in place of the transducing cytoplasmic proteins.

B. Cellular Targeting Domains

A signal peptide or sequence provides for transport of the chimericprotein to the cell surface membrane, where the same or other sequencescan encode binding of the chimeric protein to the cell surface membrane.While there is a general motif of signal sequences, two or threeN-terminal polar amino acids followed by about 15-20 primarilyhydrophobic amino acids, the individual amino acids can be widelyvaried. Therefore, substantially any signal peptide can be employedwhich is functional in the host and may or may not be associated withone of the other domains of the chimeric protein. Normally, the signalpeptide is processed and will not be retained in the mature chimericprotein. The sequence encoding the signal peptide is at the 5'-end ofthe coding sequence and will include the initiation methionine codon.

The choice of membrane retention domain is not critical to thisinvention, since it is found that such membrane retention domains aresubstantially fungible and there is no critical amino acid required forbinding or bonding to another membrane region for activation. Thus, themembrane retention domain can be isolated from any convenient surfacemembrane or cytoplasmic protein, whether endogenous to the host cell ornot.

There are at least two different membrane retention domains: atransmembrane retention domain, which is an amino acid sequence whichextends across the membrane; and a lipid membrane retention domain,which lipid associates with the lipids of the cell surface membrane.

For the most part, for ease of construction, the transmembrane domain ofthe cytoplasmic domain or the receptor domain can be employed, which maytend to simplify the construction of the fused protein. However, for thelipid membrane retention domain, the processing signal will usually beadded at the 5' end of the coding sequence for N-terminal binding to themembrane and, proximal to the 3' end for C-terminal binding. The lipidmembrane retention domain will have a lipid of from about 12 to 24carbon atoms, particularly 14 carbon atoms, more particularly myristoyl,joined to glycine. The signal sequence for the lipid binding domain isan N-terminal sequence and can be varied widely, usually having glycineat residue 2 and lysine or arginine at residue 7 (Kaplan, et al., Mol.Cell. Biol. (1988) 8, 2435). Peptide sequences involvingpost-translational processing to provide for lipid membrane binding aredescribed by Carr, et al., PNAS USA (1988) 79, 6128; Aitken, et al.,FEBS Lett. (1982) 150, 314; Henderson, et al., PNAS USA (1983) 80, 319;Schulz, et al., Virology (1984), 123, 2131; Dellman, et al., Nature(1985) 314, 374; and reviewed in Ann. Rev. of Biochem. (1988) 57, 69. Anamino acid sequence of interest includes the sequenceM-G-S-S-K-S-K-P-K-D-P-S-Q-R SEQ ID NO:1!. Various DNA sequences can beused to encode such sequence in the fused receptor protein.

Generally, the transmembrane domain will have from about 18-30 aminoacids, more usually about 20-30 amino acids, where the central portionwill be primarily neutral, non-polar amino acids, and the termini of thedomain will be polar amino acids, frequently charged amino acids,generally having about 1-2 charged, primarily basic amino acids at thetermini of the transmembrane domain followed by a helical break residue,e.g. pro- or gly-.

C. Ligand Binding Domain

The ligand binding ("dimerization") domain of a chimeric protein of thisinvention can be any convenient domain which will allow for inductionusing a natural or unnatural ligand, preferably an unnatural syntheticligand. The binding domain can be internal or external to the cellularmembrane, depending upon the nature of the construct and the choice ofligand. A wide variety of binding proteins, including receptors, areknown, including binding proteins associated with the cytoplasmicregions indicated above. Of particular interest are binding proteins forwhich ligands (preferably small organic ligands) are known or may bereadily produced. These receptors or ligand binding domains include theFKBPs and cyclophilin receptors, the steriod receptors, the tetracyclinereceptor, the other receptors indicated above, and the like, as well as"unnatural" receptors, which can be obtained from antibodies,particularly the heavy or light chain subunit, mutated sequencesthereof, random amino acid sequences obtained by stochastic procedures,combinatorial syntheses, and the like. For the most part, the receptordomains will be at least about 50 amino acids, and fewer than about 350amino acids, usually fewer than 200 amino acids, either as the naturaldomain or truncated active portion thereof. Preferably the bindingdomain will be small (<25 kDa, to allow efficient transfection in viralvectors), monomeric (this rules out the avidin-biotin system),nonimmunogenic, and should have synthetically accessible, cellpermeable, nontoxic ligands that can be configured for dimerization.

The receptor domain can be intracellular or extracellular depending uponthe design of the construct encoding the chimeric protein and theavailability of an appropriate ligand. For hydrophobic ligands, thebinding domain can be on either side of the membrane, but forhydrophilic ligands, particularly protein ligands, the binding domainwill usually be external to the cell membrane, unless there is atransport system for internalizing the ligand in a form in which it isavailable for binding. For an intracellular receptor, the construct canencode a signal peptide and transmembrane domain 5' or 3' of thereceptor domain sequence or by having a lipid attachment signal sequence5' of the receptor domain sequence. Where the receptor domain is betweenthe signal peptide and the transmembrane domain, the receptor domainwill be extracellular.

The portion of the construct encoding the receptor can be subjected tomutagenesis for a variety of reasons. The mutagenized protein canprovide for higher binding affinity, allow for discrimination by theligand of the naturally occurring receptor and the mutagenized receptor,provide opportunities to design a receptor-ligand pair, or the like. Thechange in the receptor can involve changes in amino acids known to be atthe binding site, random mutagenesis using combinatorial techniques,where the codons for the amino acids associated with the binding site orother amino acids associated with conformational changes can be subjectto mutagenesis by changing the codon(s) for the particular amino acid,either with known changes or randomly, expressing the resulting proteinsin an appropriate prokaryotic host and then screening the resultingproteins for binding. Illustrative of this situation is to modifyFKBP12's Phe36 to Ala and/or Asp37 to Gly or Ala to accommodate asubstituent at positions 9 or 10 of FK506 or FK520. In particular,mutant FKBP12 moieties which contain Val, Ala, Gly, Met or other smallamino acids in place of one or more of Tyr26, Phe36, Asp37, Tyr82 andPhe99 are of particular interest as receptor domains for FK506-type andFK-520-type ligands containing modifications at C9 and/or C10.

Antibody subunits, e.g. heavy or light chain, particularly fragments,more particularly all or part of the variable region, or fusions ofheavy and light chain to create high-affinity binding, can be used asthe binding domain. Antibodies can be prepared against haptenicmolecules which are physiologically acceptable and the individualantibody subunits screened for binding affinity. The cDNA encoding thesubunits can be isolated and modified by deletion of the constantregion, portions of the variable region, mutagenesis of the variableregion, or the like, to obtain a binding protein domain that has theappropriate affinity for the ligand. In this way, almost anyphysiologically acceptable haptenic compound can be employed as theligand or to provide an epitope for the ligand. Instead of antibodyunits, natural receptors can be employed, where the binding domain isknown and there is a useful ligand for binding.

The ability to employ in vitro mutagenesis or combinatorialmodifications of sequences encoding proteins allows for the productionof libraries of proteins which can be screened for binding affinity fordifferent ligands. For example, one can totally randomize a sequence of1 to 5, 10 or more codons, at one or more sites in a DNA sequenceencoding a binding protein, make an expression construct and introducethe expression construct into a unicellular microorganism, and develop alibrary. One can then screen the library for binding affinity to one ordesirably a plurality of ligands. The best affinity sequences which arecompatible with the cells into which they would be introduced can thenbe used as the binding domain. The ligand would be screened with thehost cells to be used to determine the level of binding of the ligand toendogenous proteins. A binding profile could be defined weighting theratio of binding affinity to the mutagenized binding domain with thebinding affinity to endogenous proteins. Those ligands which have thebest binding profile could then be used as the ligand. Phage displaytechniques, as a non-limiting example, can be used in carrying out theforegoing.

D. Multimerization

The transduced signal will normally result from ligand-mediatedoligomerization of the chimeric protein molecules, i.e. as a result ofoligomerization following ligand binding, although other binding events,for example allosteric activation, can be employed to initiate a signal.The construct of the chimeric protein will vary as to the order of thevarious domains and the number of repeats of an individual domain. Forthe extracellular receptor domain in the 5'-3' direction oftranscription, the construct will encode a protein comprising the signalpeptide, the receptor domain, the transmembrane domain and the signalinitiation domain, which last domain will be intracellular(cytoplasmic). However, where the receptor domain is intracellular,different orders may be employed, where the signal peptide can befollowed by either the receptor or signal initiation domain, followed bythe remaining domain, or with a plurality of receptor domains, thesignal initiation domain can be sandwiched between receptor domains.Usually, the active site of the signal initiation domain will beinternal to the sequence and not require a free carboxyl terminus.Either of the domains can be multimerized, particularly the receptordomain, usually having not more than about 5 repeats, more usually notmore than about 3 repeats.

For multimerizing the receptor, the ligand for the receptor domains ofthe chimeric surface membrane proteins will usually be multimeric in thesense that it will have at least two binding sites, with each of thebinding sites capable of binding to the receptor domain. Desirably, thesubject ligands will be a dimer or higher order oligomer, usually notgreater than about tetrameric, of small synthetic organic molecules, theindividual molecules typically being at least about 150 D and fewer thanabout 5 kD, usually fewer than about 3 kD. A variety of pairs ofsynthetic ligands and receptors can be employed. For example, inembodiments involving natural receptors, dimeric FK506 can be used withan FKBP receptor, dimerized cyclosporin A can be used with thecyclophilin receptor, dimerized estrogen with an estrogen receptor,dimerized glucocorticoids with a glucocorticoid receptor, dimerizedtetracycline with the tetracycline receptor, dimerized vitamin D withthe vitamin D receptor, and the like. Alternatively higher orders of theligands, e.g. trimeric can be used. For embodiments involving unnaturalreceptors, e.g. antibody subunits, modified antibody subunits ormodified receptors and the like, any of a large variety of compounds canbe used. A significant characteristic of these ligand units is that theybind the receptor with high affinity (preferably with a K_(d) ≦10⁻⁸ M)and are able to be dimerized chemically.

The ligand can have different receptor binding molecules with differentepitopes (also referred to as "HED" reagents, since they can mediatehetero-dimerization or hetero-oligomerization of chimeric proteinshaving the same or different binding domains. For example, the ligandmay comprise FK506 or an FK506-type moiety and a CsA or a cyclosporintype moiety. Both moieties are covalently attached to a common linkermoiety. Such a ligand would be useful for mediating the oligomerizationof a first and second chimeric protein where the first chimeric proteincontains a receptor domain such as an FKBP12 which is capable of bindingto the FK506-type moiety and the second chimeric protein contains areceptor domain such as cyclophilin which is capable of binding to thecyclosporin A-type moiety.

VI. Cells

The cells may be procaryotic, but are preferably eucaryotic, includingplant, yeast, worm, insect and mammalian. At present it is especiallypreferred that the cells be mammalian cells, particularly primate, moreparticularly human, but can be associated with any animal of interest,particularly domesticated animals, such as equine, bovine, murine,ovine, canine, feline, etc. Among these species, various types of cellscan be involved, such as hematopoietic, neural, mesenchymal, cutaneous,mucosal, stromal, muscle, spleen, reticuloendothelial, epithelial,endothelial, hepatic, kidney, gastrointestinal, pulmonary, etc. Ofparticular interest are hematopoietic cells, which include any of thenucleated cells which may be involved with the lymphoid ormyelomonocytic lineages. Of particular interest are members of the T-and B-cell lineages, macrophages and monocytes, myoblasts andfibroblasts. Also of particular interest are stem and progenitor cells,such as hematopoietic neural, stromal, muscle, hepatic, pulmonary,gastrointestinal, etc.

The cells can be autologous cells, syngeneic cells, allogenic cells andeven in some cases, xenogeneic cells. The cells may be modified bychanging the major histocompatibility complex ("MHC") profile, byinactivating β₂ -microglobulin to prevent the formation of functionalClass I MHC molecules, inactivation of Class II molecules, providing forexpression of one or more MHC molecules, enhancing or inactivatingcytotoxic capabilities by enhancing or inhibiting the expression ofgenes associated with the cytotoxic activity, or the like.

In some instances specific clones or oligoclonal cells may be ofinterest, where the cells have a particular specificity, such as T cellsand B cells having a specific antigen specificity or homing target sitespecificity.

VII. Ligands

A wide variety of ligands, including both naturally occurring andsynthetic substances, can be used in this invention to effectoligomerization of the chimeric protein molecules. Applicable andreadily observable or measurable criteria for selecting a ligand are:(A) the ligand is physiologically acceptable (i.e., lacks undue toxicitytowards the cell or animal for which it is to be used), (B) it has areasonable therapeutic dosage range, (C) desirably (for applications inwhole animals, including gene therapy applications), it can be takenorally (is stable in the gastrointestinal system and absorbed into thevascular system), (D) it can cross the cellular and other membranes, asnecessary, and (E) binds to the receptor domain with reasonable affinityfor the desired application. A first desirable criterion is that thecompound is relatively physiologically inert, but for its activatingcapability with the receptors. The less the ligand binds to nativereceptors and the lower the proportion of total ligand which binds tonature receptors, the better the response will normally be.Particularly, the ligand should not have a strong biological effect onnative proteins. For the most part, the ligands will be non-peptide andnon-nucleic acid.

The subject compounds will for the most part have two or more units,where the units can be the same or different, joined together through acentral linking group. The "units" will be individual moieties (e.g.,FK506, FK520, cyclosporin A, a steroid, etc.) capable of binding thereceptor domain. Each of the units will usually be joined to the linkinggroup through the same reactive moieties, at least in homodimers orhigher order homo-oligomers.

As indicated above, there are a variety of naturally-occurring receptorsfor small non-proteinaceous organic molecules, which small organicmolecules fulfill the above criteria, and can be dimerized at varioussites to provide a ligand according to the subject invention.Substantial modifications of these compounds are permitted, so long asthe binding capability is retained and with the desired specificity.Many of the compounds will be macrocyclics, e.g. macrolides. Suitablebinding affinities will be reflected in Kd values well below 10⁻⁴,preferably below 10⁻⁶, more preferably below about 10⁻⁷, althoughbinding affinities below 10⁻⁹ or 10⁻¹⁰ are possible, and in some caseswill be most desirable.

Currently preferred ligands comprise oligomers, usually dimers, ofcompounds capable of binding to an FKBP protein and/or to a cyclophilinprotein. Such ligands includes homo- and heteromultimers (usually 2-4,more usually 2-3 units) of cyclosporin A, FK506, FK520, and rapamycin,and derivatives thereof, which retain their binding capability to thenatural or mutagenized binding domain. Many derivatives of suchcompounds are already known, including synthetic high affinity FKBPligands, which can be used in the practice of this invention. See e.g.Holt et al, J Am Chem Soc 1993,115, 9925-9935. Sites of interest forlinking of FK506 and analogs thereof include positions involving annularcarbon atoms from about 17 to 24 and substituent positions bound tothose annular atoms, e.g. 21 (allyl), 22, 37, 38, 39 and 40, or 32(cyclohexyl), while the same positions except for 21 are of interest forFK520. For cyclosporin, sites of interest include MeBmt, position 3 andposition 8.

Of particular interest are modifications to the ligand which change itsbinding characteristics, particularly with respect to the ligand'snaturally occurring receptor. Concomitantly, one would change thebinding protein to accommodate the change in the ligand. For example,one can modify the groups at position 9 or 10 of FK506 (see Van Duyne etal (1991) Science 252, 839), so as to increase their steric requirement,by replacing the hydroxyl with a group having greater stericrequirements, or by modifying the carbonyl at position 10, replacing thecarbonyl with a group having greater steric requirements orfunctionalizing the carbonyl, e.g. forming an N-substituted Schiff'sbase or imine, to enhance the bulk at that position. Variousfunctionalities which can be conveniently introduced at those sites arealkyl groups to form ethers, acylamido groups, N-alkylated amines, wherea 2-hydroxyethylimine can also form a 1,3-oxazoline, or the like.Generally, the substituents will be from about 1 to 6, usually 1 to 4,and more usually 1 to 3 carbon atoms, with from 1 to 3, usually 1 to 2heteroatoms, which will usually be oxygen, sulfur, nitrogen, or thelike. By using different derivatives of the basic structure, one cancreate different ligands with different conformational requirements forbinding. By mutagenizing receptors, one can have different receptors ofsubstantially the same sequence having different affinities for modifiedligands not differing significantly in structure.

Other ligands which can be used are steroids. The steroids can beoligomerized, so that their natural biological activity is substantiallydiminished without loss of their binding capability with respect to achimeric protein containing one or more steroid receptor domains. By wayof non-limiting example, glucocorticoids and estrogens can be so used.Various drugs can also be used, where the drug is known to bind to aparticular receptor with high affinity. This is particularly so wherethe binding domain of the receptor is known, thus permitting the use inchimeric proteins of this invention of only the binding domain, ratherthan the entire native receptor protein. For this purpose, enzymes andenzyme inhibitors can be used.

A. Linkers

Various functionalities can be involved in the linking, such as amidegroups, including carbonic acid derivatives, ethers, esters, includingorganic and inorganic esters, amino, or the like. To provide forlinking, the particular monomer can be modified by oxidation,hydroxylation, substitution, reduction, etc., to provide a site forcoupling. Depending on the monomer, various sites can be selected as thesite of coupling.

The multimeric ligands can be synthesized by any convenient means, wherethe linking group will be at a site which does not interfere with thebinding of the binding site of a ligand to the receptor. Where theactive site for physiological activity and binding site of a ligand tothe receptor domain are different, it will usually be desirable to linkat the active site to inactivate the ligand. Various linking groups canbe employed, usually of from 1-30, more usually from about 1-20 atoms inthe chain between the two molecules (other than hydrogen), where thelinking groups will be primarily composed of carbon, hydrogen, nitrogen,oxygen, sulphur and phosphorous. The linking groups can involve a widevariety of functionalities, such as amides and esters, both organic andinorganic, amines, ethers, thioethers, disulfides, quaternary ammoniumsalts, hydrazines, etc. The chain can include aliphatic, alicyclic,aromatic or heterocyclic groups. The chain will be selected based onease of synthesis and the stability of the multimeric ligand. Thus, ifone wishes to maintain long-term activity, a relatively inert chain willbe used, so that the multimeric ligand link will not be cleaved.Alternatively, if one wishes only a short half-life in the blood stream,then various groups can be employed which are readily cleaved, such asesters and amides, particularly peptides, where circulating and/orintracellular proteases can cleave the linking group.

Various groups can be employed as the linking group between ligands,such as alkylene, usually of from 2 to 20 carbon atoms, azalkylene(where the nitrogen will usually be between two carbon atoms), usuallyof from 4 to 18 carbon atoms), N-alkylene azalkylene (see above),usually of from 6 to 24 carbon atoms, arylene, usually of from 6 to 18carbon atoms, ardialkylene, usually of from 8 to 24 carbon atoms,bis-carboxamido alkylene of from about 8 to 36 carbon atoms, etc.Illustrative groups include decylene, octadecylene, 3-azapentylene,5-azadecylene, N-butylene 5-azanonylene, phenylene, xylylene,p-dipropylenebenzene, bis-benzoyl 1,8-diaminooctane and the like.Multivalent or other (see below) ligand molecules containing linkermoieties as described above can be evaluated with chimeric proteins ofthis invention bearing corresponding receptor domains using materialsand methods described in the examples which follow.

B. Ligand Characteristics

For intracellular binding domains, the ligand will be selected to beable to be transferred across the membrane in a bioactive form, that is,it will be membrane permeable. Various ligands are hydrophobic or can bemade so by appropriate modification with lipophilic groups.Particularly, the linking bridge can serve to enhance the lipophilicityof the ligand by providing aliphatic side chains of from about 12 to 24carbon atoms. Alternatively, one or more groups can be provided whichwill enhance transport across the membrane, desirably without endosomeformation.

In some instances, multimeric ligands need not be employed. For example,molecules can be employed where two different binding sites provide fordimerization of the receptor. In other instances, binding of the ligandcan result in a conformational change of the receptor domain, resultingin activation, e.g. oligomerization, of the receptor. Other mechanismsmay also be operative for inducing the signal, such as binding a singlereceptor with a change in conformation resulting in activation of thecytoplasmic domain.

C. Ligand Antagonists

Monomeric ligands can be used for reversing the effect of the multimericligand, i.e., for inhibiting or disrupting oligomer formation ormaintenance. Thus, if one wishes to rapidly terminate the effect ofcellular activation, a monomeric ligand can be used. Conveniently, theparent ligand moiety can be modified at the same site as the multimer,using the same procedure, except substituting a monofunctional compoundfor the polyfunctional compound. Instead of the polyamines, monoamines,particularly of from 2 to 20 (although they can be longer), and usually2 to 12, carbon atoms can be used, such as ethylamine, hexylamine,benzylamine, etc. Alternatively, the monovalent parent compound can beused, in cases in which the parent compound does not have undueundesirable physiological activity (e.g. immunosuppression, mitogenesis,toxicity, etc.)

D. Illustrative Hetero-oligomerizing (HED) and Homo-oligomerizing (HOD)Reagents With "bumps" That Can Bind to Mutant Receptors ContainingCompensatory Mutations

As discussed above, one can prepare modified HED/HOD reagents that willfail to bind appreciably to their wildtype receptors (e.g., FKBP12) dueto the presence of substituents ("bumps") on the reagents thatsterically clash with sidechain residues in the receptor's bindingpocket. One may also make corresponding receptors that contain mutationsat the interfering residues ("compensatory mutations") and thereforegain the ability to bind ligands with bumps. Using "bumped" ligandmoieties and receptor domains bearing compensatory mutations shouldenhance the specificity and thus the potency of our reagents. Bumpedreagents should not bind to the endogenous, wildtype receptors, whichcan otherwise act as a "buffer" toward dimerizers based on naturalligand moieties. In addition, the generation of novel receptor-ligandpairs should simultaneously yield the HED reagents that will be usedwhen heterodimerization is required. For example, regulated vesiclefusion may be achieved by inducing the heterodimerization of syntaxin (aplasma membrane fusion protein) and synaptobrevin (a vesicle membranefusion protein) using a HED reagent. This would not only provide aresearch tool, but could also serve as the basis of a gene therapytreatment for diabetes, using appropriately modified secretory cells.

As an illustration of "Bumped FK1012s" we prepared C10 acetamide andformamide derivatives of FK506. See FIG. 16A and our report, Spencer etal, "Controlling Signal Transduction with Synthetic Ligands," Science262 5136 (1993): 1019-1024 for additional details concerning thesyntheses of FK1012s A-C and FK506M. We chose to create two classes ofbumped FK1012s: one with a bump at C10 and one at C9. The R- andS-isomers of the C10 acetamide and formamide of FK506 have beensynthesized according to the reaction sequence in FIG. 05B. These bumpedderivatives have lost at least three orders of magnitude in theirbinding affinity towards FKBP12 (FIG. 16A (panel B)). The affinitieswere determined by measuring the ability of the derivatives to inhibitFKBP12's rotamase activity.

An illustrative member of a second class of C9-bumped derivatives is thespiro-epoxide (depicted in FIG. 16B (panel C)), which has been preparedby adaptation of known procedures. See e.g. Fisher et al, J Org Chem 568(1991): 2900-7 and Edmunds et al, Tet Lett 32 48 (1991):819-820. Aparticularly interesting series of C9 derivatives are characterized bytheir sp3 hybridization and reduced oxidation state at C9. Several suchcompounds have been synthesized according to the reactions shown in FIG.16C.

It should be appreciated that heterodimers (and otherhetero-oligomerizers) must be constructed differently than thehomodimers, at least for applications where homodimer contaminationcould adversely affect their successful use. One illustrative syntheticstrategy developed to overcome this problem is outlined in FIG. 16B(panel D). Coupling of mono alloc-protected 1,6-hexanediamine (Stahl etal, J Org Chem 43 11 (1978): 2285-6) with a derivatized form of FK506 inmethylene chloride with an excess of triethylamine gave analloc-amine-substituted FK506 in 44% yield. This intermediate can now beused in the coupling with any activated FK506 (or bumped-FK506)molecule. Deprotection with catalytic tetrakis-triphenylphosphinepalladium in the presence of dimedone at rt in THF removes the amineprotecting group. Immediate treatment with an activated FK506derivative, followed by desilylation leads to a dimeric product. Thistechnique has been used to synthesize the illustrated HOD and HEDreagents.

E. Illustrative Cyclosporin-Based Reagents

Cyclosporin A (CsA) is a cyclic undecapeptide that binds with highaffinity (6 nM) to its intracellular receptor cyclophilin, an 18 kDamonomeric protein. The resulting complex, like the FKBP12-FK506 complex,binds to and inactivates the protein phosphatase calcineurin resultingin the immunosuppressive properties of the drug. As a furtherillustration of this invention, we have dimerized CsA via its MeBmt1sidechain in 6 steps and 35% overall yield to give (CsA)2 (FIG. 17,steps 1-4 were conducted as reported in Eberle et al, J Org Chem 57 9(1992): 2689-91). As with FK1012s, the site for dimerization was chosensuch that the resulting dimer can bind to two molecules of cyclophilinyet cannot bind to calcineurin following cyclophilin-binding. We havedemonstrated that (CsA)2 binds to cyclophilin A with 1:2 stoichiometry.Hence, (CsA)2, like FK1012s, does not inhibit signaling pathways and isthus neither immunosuppressive nor toxic.

VIII. Target Gene

A. Transcription Initiation Region

The second construct or second series of constructs will have aresponsive element in the 5' region, which responds to ligand-mediatedoligomerization of the chimeric receptor protein, presumably via thegeneration and transduction of a transcription initiation signal asdiscussed infra. Therefore, it will be necessary to know at least onetranscription initiation system, e.g. factor, which is activated eitherdirectly or indirectly, by the cytoplasmic domain or can be activated byassociation of two domains. It will also be necessary to know at leastone promoter region which is responsive to the resulting transcriptioninitiation system. Either the promoter region or the gene under itstranscriptional control need be known. In other words, an action domaincan be selected for the chimeric proteins (encoded by a "first" seriesconstruct) based on the role of that action domain in initiatingtranscription via a given promoter or responsive element. See e.g.Section V(A) "Cytoplasmic domains", above.

Where the responsive element is known, it can be included in the targetgene construct to provide an expression cassette for integration intothe genome (whether episomally or by chromosomal incorporation). It isnot necessary to have isolated the particular sequence of the responsiveelement, so long as a gene is known which is transcriptionally activatedby the cytoplasmic domain upon natural ligand binding to the proteincomprising the cytoplasmic domain. Homologous recombination could thenbe used for insertion of the gene of interest downstream from thepromoter region to be under the transcriptional regulation of theendogenous promoter region. Where the specific responsive elementsequence is known, that can be used in conjunction with a differenttranscription initiation region, which can have other aspects, such as ahigh or low activity as to the rate of transcription, binding ofparticular transcription factors and the like.

The expression construct will therefore have at its 5' end in thedirection of transcription, the responsive element and the promotersequence which allows for induced transcription initiation of a targetgene of interest, usually a therapeutic gene. The transcriptionaltermination region is not as important, and can be used to enhance thelifetime of or make short half-lived mRNA by inserting AU sequenceswhich serve to reduce the stability of the mRNA and, therefore, limitthe period of action of the protein. Any region can be employed whichprovides for the necessary transcriptional termination, and asappropriate, translational termination.

The responsive element can be a single sequence or can be oligomerized,usually having not more than about 5 repeats, usually having about 3repeats.

Homologous recombination can also be used to remove or inactivateendogenous transcriptional control sequences, including promoter and/orresponsive elements, which are responsive to the oligomerization event,and/or to insert such responsive transcriptional control sequencesupstream of a desired endogenous gene.

B. Product

A wide variety of genes can be employed as the target gene, includinggenes that encode a protein of interest or an antisense sequence ofinterest or a ribozyme of interest. The target gene can be any sequenceof interest which provides a desired phenotype. The target gene canexpress a surface membrane protein, a secreted protein, a cytoplasmicprotein, or there can be a plurality of target genes which can expressdifferent types of products. The target gene may be an antisensesequence which can modulate a particular pathway by inhibiting atranscriptional regulation protein or turn on a particular pathway byinhibiting the translation of an inhibitor of the pathway. The targetgene can encode a ribozyme which may modulate a particular pathway byinterfering, at the RNA level, with the expression of a relevanttranscriptional regulator or with the expression of an inhibitor of aparticular pathway. The proteins which are expressed, singly or incombination, can involve homing, cytotoxicity, proliferation, immuneresponse, inflammatory response, clotting or dissolving of clots,hormonal regulation, or the like. The proteins expressed could benaturally-occurring, mutants of naturally-occurring proteins, uniquesequences, or combinations thereof.

The gene can be any gene which is secreted by a cell, so that theencoded product can be made available at will, whenever desired orneeded by the host. Various secreted products include hormones, such asinsulin, human growth hormone, glucagon, pituitary releasing factor,ACTH, melanotropin, relaxin, etc.; growth factors, such as EGF, IGF-1,TGF-α, -β, PDGF, G-CSF, M-CSF, GM-CSF, FGF, erythropoietin,megakaryocytic stimulating and growth factors, etc.; interleukins, suchas IL-1 to -13; TNF-α and -β, etc.; and enzymes, such as tissueplasminogen activator, members of the complement cascade, performs,superoxide dismutase, coagulation factors, antithrombin-III, FactorVIIIc, Factor VIIIvW, α-anti-trypsin, protein C, protein S, endorphins,dynorphin, bone morphogenetic protein, CFTR, etc.

The gene can be any gene which is naturally a surface membrane proteinor made so by introducing an appropriate signal peptide andtransmembrane sequence. Various proteins include homing receptors, e.g.L-selectin (Mel-14), blood-related proteins, particularly having akringle structure, e.g. Factor VIIIc, Factor VIIIvW, hematopoietic cellmarkers, e.g. CD3, CD4, CD8, B cell receptor, TCR subunits α, β, γ, δ,CD10, CD19, CD28, CD33, CD38, CD41, etc., receptors, such as theinterleukin receptors IL-2R, IL-4R, etc., channel proteins, for influxor efflux of ions, e.g. H⁺, Ca⁺², K⁺, Na⁺, Cl⁻, etc., and the like;CFTR, tyrosine activation motif, ζ activation protein, etc.

Proteins may be modified for transport to a vesicle for exocytosis. Byadding the sequence from a protein which is directed to vesicles, wherethe sequence is modified proximal to one or the other terminus, orsituated in an analogous position to the protein source, the modifiedprotein will be directed to the Golgi apparatus for packaging in avesicle. This process in conjunction with the presence of the chimericproteins for exocytosis allows for rapid transfer of the proteins to theextracellular medium and a relatively high localized concentration.

Also, intracellular proteins can be of interest, such as proteins inmetabolic pathways, regulatory proteins, steroid receptors,transcription factors, etc., particularly depending upon the nature ofthe host cell. Some of the proteins indicated above can also serve asintracellular proteins.

The following are a few illustrations of different genes. In T-cells,one may wish to introduce genes encoding one or both chains of a T-cellreceptor. For B-cells, one could provide the heavy and light chains foran immunoglobulin for secretion. For cutaneous cells, e.g.keratinocytes, particularly stem cells keratinocytes, one could providefor infectious protection, by secreting α-, β- or -γ interferon,antichemotactic factors, proteases specific for bacterial cell wallproteins, etc.

In addition to providing for expression of a gene having therapeuticvalue, there will be many situations where one may wish to direct a cellto a particular site. The site can include anatomical sites, such aslymph nodes, mucosal tissue, skin, synovium, lung or other internalorgans or functional sites, such as clots, injured sites, sites ofsurgical manipulation, inflammation, infection, etc. By providing forexpression of surface membrane proteins which will direct the host cellto the particular site by providing for binding at the host target siteto a naturally-occurring epitope, localized concentrations of a secretedproduct can be achieved. Proteins of interest include homing receptors,e.g. L-selectin, GMP140, CLAM-1, etc., or addressing, e.g. ELAM-1, PNAd,LNAd, etc., clot binding proteins, or cell surface proteins that respondto localized gradients of chemotactic factors. There are numeroussituations where one would wish to direct cells to a particular site,where release of a therapeutic product could be of great value.

In many situations one may wish to be able to kill the modified cells,where one wishes to terminate the treatment, the cells becomeneoplastic, in research where the absence of the cells after theirpresence is of interest, or other event. For this purpose one canprovide for the expression of the Fas antigen or TNF receptor fused to abinding domain. (Watanable-Fukunaga et al. Nature (1992) 356, 314-317)In the original modification, one can provide for constitutiveexpression of such constructs, so that the modified cells have suchproteins on their surface or present in their cytoplasm. Alternatively,one can provide for controlled expression, where the same or differentligand can initiate expression and initiate apoptosis. By providing forthe cytoplasmic portions of the Fas antigen or TNF receptor in thecytoplasm joined to binding regions different from the binding regionsassociated with expression of a target gene of interest, one can killthe modified cells under controlled conditions.

C. Illustrative Exemplifications

By way of illustration, cardiac patients or patients susceptible tostroke may be treated as follows. Cells modified as described herein maybe administered to the patient and retained for extended periods oftime. Illustrative cells include plasma cells, B-cells, T-cells, orother hematopoietic cells. The cell would be modified to express aprotein which binds to a blood clot, e.g. having a kringle domainstructure or an adhesive interactive protein, e.g. CD41, and to expressa clot dissolving protein, e.g. tissue plasminogen activator,streptokinase, etc. In this way, upon ligand-mediated oligomerization,the cells would accumulate at the site of the clot and provide for ahigh localized concentration of the thrombolytic protein.

Another example is reperfusion injury. Cells of limited lifetime couldbe employed, e.g. macrophages or polymorphonuclear leukocytes("neutrophils"). The cells would have a neutrophil homing receptor todirect the cells to a site of reperfusion injury. The cell would alsoexpress superoxide dismutase, to destroy singlet oxygen and inhibitradical attack on the tissue.

A third example is autoimmune disease. Cells of extended lifetime, e.g.T cells could be employed. The constructs would provide for a homingreceptor for homing to the site of autoimmune injury and for cytotoxicattack on cells causing the injury. The therapy would then be directedagainst cells causing the injury. Alternatively, one could provide forsecretion of soluble receptors or other peptide or protein, where thesecretion product would inhibit activation of the injury causing cellsor induce anergy. Another alternative would be to secrete anantiinflammatory product, which could serve to diminish the degenerativeeffects.

A fourth example involves treatment of chronic pain with endorphin viaencapsulation. A stock of human fibroblasts is transfected with aconstruct in which the chimeric transcriptional regulatory proteincontrols the transcription of human endorphin. The DNA constructconsists of three copies of the binding site for the HNF-1*transcription factor GTTAAGTTAAC SEQ ID NO:2! upstream of a TATAAA siteand a transcriptional initiation site. The endorphin cDNA would beinserted downstream of the initiation site and upstream of apolyadenylation and termination sequences. Optionally, the endorphincDNA is outfitted with "PEST" sequences to make the protein unstable orAUUA sequences in the 3' nontranslated region of the MRNA to allow it tobe degraded quickly.

The fibroblasts are also transfected with a construct having twotranscription units, one of which would encode the HNF-1* cDNA truncatedto encode just the DNA binding sequences from amino acids 1 to 250coupled to a trimeric FKBP binding domain under the transcriptional andtranslational control of regulatory initiation and termination regionsfunctional in the fibroblasts. The construct would include an additionaltranscription unit driven by the same regulatory regions directing theproduction of a transcriptional activation domain derived from HNF-4coupled to trimeric FKBP'. (The prime intends an altered FKBP that bindsat nM concentration to a modified FK506. The modification inhibitsbinding to the endogenous FKBP.)

These genetically modified cells would be encapsulated to inhibit immunerecognition and placed under the patient's skin or other convenientinternal site. When the patient requires pain medication, the patientadministers a dimeric ligand FK506-FK506', where about 1 μg to 1 mgwould suffice. In this manner one could provide pain relief withoutinjections or the danger of addiction.

A fifth example is the treatment of osteoporosis. Lymphocytes can beclonally developed or skin fibroblasts grown in culture from the patientto be treated. The cells would be transfected as described above, wherea bone morphogenic factor cDNA gene would replace the endorphin gene.For lymphocytes, antigen specific clones could be used which would allowtheir destruction with antibodies to the idiotype of the slg. Inaddition, administration of the antigen for the slg would expand thecell population to increase the amount of the protein which could bedelivered. The lymphocyte clones would be infused and the ligandadministered as required for production of the bone morphogenic factor.By monitoring the response to the ligand, one could adjust the amount ofbone morphogenic factor which is produced, so as to adjust the dosage tothe required level.

A sixth situation has general application in conjunction with genetherapies involving cells which may be required to be destroyed. Forexample, a modified cell may become cancerous or result in anotherpathologic state. Constructs would be transfected into the modifiedcells having the necessary transcriptional and translational regulatoryregions and encoding a protein which upon oligomerization results incell death, e.g. apoptosis. For example, the fas antigen or Apo-1antigen induces apoptosis in most cell types (Trauth et al. (1989)Science 245, 301-305; Watanaba-Fukunaga et al. (1992) Nature 356, 314)In this manner by co-transfecting the protective constructs into cellsused for gene therapy or other purpose, where there may be a need toensure the death of a portion or all of the cells, the cells may bemodified to provide for controlled cytotoxicity by means of the ligand.

Another situation is to modify antigen specific T cells, where one canactivate expression of a protein product to activate the cells. The Tcell receptor could be directed against tumor cells, pathogens, cellsmediating autoimmunity, and the like. By providing for activation of thecells, for example, an interleukin such as IL-2, one could provide forexpansion of the modified T cells in response to a ligand. Other uses ofthe modified T cells would include expression of homing receptors fordirecting the T cells to specific sites, where cytotoxicity,upregulation of a surface membrane protein of target cells, e.g.endothelial cells, or other biological event would be desired.

Alternatively one may want to deliver high doses of cytotoxic factors tothe target site. For example, upon recognition of tumor antigens via ahoming receptor, tumor-infiltrating lymphocytes (TILs) may be triggeredto deliver toxic concentrations of TNF or other similar product.

Another alternative is to export hormones or factors which areexocytosed. By providing for enhanced exocytosis, a greater amount ofthe hormone or factor will be exported; in addition, if there is afeedback mechanism based on the amount of the hormone or factor in thecytoplasm, increased production of the hormone or factor will result.Or, one may provide for induced expression of the hormone or factor, sothat expression and export may be induced concomitantly.

One may also provide for proteins in retained body fluids, e.g. vascularsystem, lymph system, cerebrospinal fluid, etc. By modifying cells whichcan have an extended lifetime in the host, e.g. hematopoietic cells,keratinocytes, muscle cells, etc. particularly, stem cells, the proteinscan be maintained in the fluids for extended periods of time. The cellsmay be modified with constructs which provide for secretion orendocytosis. The constructs for secretion would have as thetranslocation domain, a signal peptide, and then as in the case of theother chimeric proteins, a binding domain and an action domain. Theaction domains may be derived from the same or different proteins. Forexample, with tissue plasminogen activator, one could have the clotbinding region as one action domain and the plasminogen active site as adifferent action domain. Alternatively, one could provide enhancedblockage of homing, by having a binding protein, such as LFA-1 as oneaction domain and a selection as a second action domain. By modifyingsubunits of proteins, e.g. integrins, T-cell receptor, slg, or the like,one could provide soluble forms of surface membrane proteins which couldbe brought together to bind to a molecule. Other opportunities arecomplement proteins, platelet membrane proteins involved in clotting,autoantigens on the surface of cells, and pathogenic molecules on thesurface of infectious agents.

IX. Introduction of Constructs into Cells

The constructs can be introduced as one or more DNA molecules orconstructs, where there will usually be at least one marker and theremay be two or more markers, which will allow for selection of host cellswhich contain the construct(s). The constructs can be prepared inconventional ways, where the genes and regulatory regions may beisolated, as appropriate, ligated, cloned in an appropriate cloninghost, analyzed by restriction or sequencing, or other convenient means.Particularly, using PCR, individual fragments including all or portionsof a functional unit may be isolated, where one or more mutations may beintroduced using "primer repair", ligation, in vitro mutagensis, etc. asappropriate. The construct(s) once completed and demonstrated to havethe appropriate sequences may then be introduced into the host cell byany convenient means. The constructs may be integrated and packaged intonon-replicating, defective viral genomes like Adenovirus,Adeno-associated virus (AAV), or Herpes simplex virus (HSV) or others,including retroviral vectors, for infection or transduction into cells.The constructs may include viral sequences for transfection, if desired.Alternatively, the construct may be introduced by fusion,electroporation, biolistics, transfection, lipofection, or the like. Thehost cells will usually be grown and expanded in culture beforeintroduction of the construct(s), followed by the appropriate treatmentfor introduction of the construct(s) and integration of theconstruct(s). The cells will then be expanded and screened by virtue ofa marker present in the construct. Various markers which may be usedsuccessfully include hprt, neomycin resistance, thymidine kinase,hygromycin resistance, etc.

In some instances, one may have a target site for homologousrecombination, where it is desired that a construct be integrated at aparticular locus. For example, one can knock-out an endogenous gene andreplace it (at the same locus or elswhere) with the gene encoded for bythe construct using materials and methods as are known in the art forhomologous recombination. Alternatively, instead of providing a gene,one may modify the transcriptional initiation region of an endogenousgene to be responsive to the signal initiating domain. In suchembodiments, transcription of an endogenous gene such as EPO, tPA, SOD,or the like, would be controlled by administration of the ligand. Forhomologous recombination, one may use either Ω or O-vectors. See, forexample, Thomas and Capecchi, Cell (1987) 51, 503-512; Mansour, et al.,Nature (1988) 336, 348-352; and Joyner, et al., Nature (1989) 338,153-156.

The constructs may be introduced as a single DNA molecule encoding allof the genes, or different DNA molecules having one or more genes. Theconstructs may be introduced simultaneously or consecutively, each withthe same or different markers. In an illustrative example, one constructwould contain a therapeutic gene under the control of a specificresponsive element (e.g. NFAT), another encoding the receptor fusionprotein comprising the signaling region fused to the ligand receptordomain (e.g. as in MZF3E). A third DNA molecule encoding a homingreceptor or other product that increases the efficiency of delivery ofthe therapeutic product may also be introduced.

Vectors containing useful elements such as bacterial or yeast origins ofrepliation, selectable and/or amplifiable markers, promoter/enhancerelements for expression in procaryotes or eucaryotes, etc. which may beused to prepare stocks of construct DNAs and for carrying outtransfections are well known in the art, and many are commerciallyavailable.

X. Administration of Cells and Ligands

The cells which have been modified with the DNA constructs are thengrown in culture under selective conditions and cells which are selectedas having the construct may then be expanded and further analyzed,using, for example, the polymerase chain reaction for determining thepresence of the construct in the host cells. Once the modified hostcells have been identified, they may then be used as planned, e.g. grownin culture or introduced into a host organism.

Depending upon the nature of the cells, the cells may be introduced intoa host organism, e.g. a mammal, in a wide variety of ways. Hematopoieticcells may be administered by injection into the vascular system, therebeing usually at least about 10⁴ cells and generally not more than about10¹⁰, more usually not more than about 10⁸ cells. The number of cellswhich are employed will depend upon a number of circumstances, thepurpose for the introduction, the lifetime of the cells, the protocol tobe used, for example, the number of administrations, the ability of thecells to multiply, the stability of the therapeutic agent, thephysiologic need for the therapeutic agent, and the like. Alternatively,with skin cells which may be used as a graft, the number of cells woulddepend upon the size of the layer to be applied to the burn or otherlesion. Generally, for myoblasts or fibroblasts, the number of cellswill at least about 10⁴ and not more than about 10⁸ and may be appliedas a dispersion, generally being injected at or near the site ofinterest. The cells will usually be in a physiologically-acceptablemedium.

Instead of ex vivo modification of the cells, in many situations one maywish to modify cells in vivo. For this purpose, various techniques havebeen developed for modification of target tissue and cells in vivo. Anumber of virus vectors have been developed, such as adenovirus andretroviruses, which allow for transfection and random integration of thevirus into the host. See, for example, Dubensky et al. (1984) Proc.Natl. Acad. Sci. USA 81, 7529-7533; Kaneda et al., (1989) Science243,375-378; Hiebert et al. (1989) Proc. Natl. Acad. Sci. USA 86,3594-3598; Hatzoglu et al. (1990) J. Biol. Chem. 265, 17285-17293 andFerry, et al. (1991) Proc. Natl. Acad. Sci. USA 88, 8377-8381. Thevector may be administered by injection, e.g. intravascularly orintramuscularly, inhalation, or other parenteral mode.

In accordance with in vivo genetic modification, the manner of themodification will depend on the nature of the tissue, the efficiency ofcellular modification required, the number of opportunities to modifythe particular cells, the accessibility of the tissue to the DNAcomposition to be introduced, and the like. By employing an attenuatedor modified retrovirus carrying a target transcriptional initiationregion, if desired, one can activate the virus using one of the subjecttranscription factor constructs, so that the virus may be produced andtransfect adjacent cells.

The DNA introduction need not result in integration in every case. Insome situations, transient maintenance of the DNA introduced may besufficient. In this way, one could have a short term effect, where cellscould be introduced into the host and then turned on after apredetermined time, for example, after the cells have been able to hometo a particular site.

The ligand providing for activation of the cytoplasmic domain may thenbe administered as desired. Depending upon the binding affinity of theligand, the response desired, the manner of administration, thehalf-life, the number of cells present, various protocols may beemployed. The ligand may be administered parenterally or orally. Thenumber of administrations will depend upon the factors described above.The ligand may be taken orally as a pill, powder, or dispersion;bucally; sublingually; injected intravascularly, intraperitoneally,subcutaneously; by inhalation, or the like. The ligand (and monomericcompound) may be formulated using conventional methods and materialswell known in the art for the various routes of administration. Theprecise dose and particular method of administration will depend uponthe above factors and be determined by the attending physician or humanor animal healthcare provider. For the most part, the manner ofadministration will be determined empirically.

In the event that the activation by the ligand is to be reversed, themonomeric compound may be administered or other single binding sitecompound which can compete with the ligand. Thus, in the case of anadverse reaction or the desire to terminate the therapeutic effect, themonomeric binding compound can be administered in any convenient way,particularly intravascularly, if a rapid reversal is desired.Alternatively, one may provide for the presence of an inactivationdomain with a DNA binding domain, or apoptosis by having Fas or TNFreceptor present as constitutively expressed constructs.

The particular dosage of the ligand for any application may bedetermined in accordance with the procedures used for therapeutic dosagemonitoring, where maintenance of a particular level of expression isdesired over an extended period of times, for example, greater thanabout two weeks, or where there is repetitive therapy, with individualor repeated doses of ligand over short periods of time, with extendedintervals, for example, two weeks or more. A dose of the ligand within apredetermined range would be given and monitored for response, so as toobtain a time-expression level relationship, as well as observingtherapeutic response. Depending on the levels observed during the timeperiod and the therapeutic response, one could provide a larger orsmaller dose the next time, following the response. This process wouldbe iteratively repeated until one obtained a dosage within thetherapeutic range. Where the ligand is chronically administered, oncethe maintenance dosage of the ligand is determined, one could then doassays at extended intervals to be assured that the cellular system isproviding the appropriate response and level of the expression product.

It should be appreciated that the system is subject to many variables,such as the cellular response to the ligand, the efficiency ofexpression and, as appropriate, the level of secretion, the activity ofthe expression product, the particular need of the patient, which mayvary with time and circumstances, the rate of loss of the cellularactivity as a result of loss of cells or expression activity ofindividual cells, and the like. Therefore, it is expected that for eachindividual patient, even if there were universal cells which could beadministered to the population at large, each patient would be monitoredfor the proper dosage for the individual.

The subject methodology and compositions may be used for the treatmentof a wide variety of conditions and indications. For example, B- andT-cells may be used in the treatment of cancer, infectious diseases,metabolic deficiencies, cardiovascular disease, hereditary coagulationdeficiencies, autoimmune diseases, joint degenerative diseases, e.g.arthritis, pulmonary disease, kidney disease, endocrine abnormalities,etc. Various cells involved with structure, such as fibroblasts andmyoblasts, may be used in the treatment of genetic deficiencies, such asconnective tissue deficiencies, arthritis, hepatic disease, etc.Hepatocytes could be used in cases where large amounts of a protein mustbe made to complement a deficiency or to deliver a therapeutic productto the liver or portal circulation.

The following examples are offered by way illustration and not by waylimitation.

EXAMPLES

Cellular Transformations and Evaluation

Example 1

Induction of Isolated IL-2 Enhancer-Binding Transcription Factors byCross-Linking the CD3 Chain of the T-Cell Receptor

The plasmid pSXNeo/IL2 (IL2-SX) (FIG. 1), which contains the placentalsecreted alkaline phosphatase gene under the control of human IL-2promoter (-325 to +47; MCB(86) 6, 3042), and related plasmid variants(i.e. NFAT-SX, NF B-SX, OAP/Oct1-SX, and AP-1-SX) in which the reportergene is under the transcriptional control of the minimal IL-2 promoter(-325 to -294 and -72 to +47) combined with synthetic oligomerscontaining various promoter elements (i.e. NFAT, NK B, OAP/Oct-1, andAP1, respectively), were made by three piece ligations of 1) pPL/SEAP(Berger, et al., Gene (1988) 66,1) cut with SspI and HindIII; 2)pSV2/Neo (Southern and Berg, J. Mol. Appl. Genet. (1982) 1, 332) cutwith NdeI, blunted with Klenow, then cut with PvuI; and 3) variouspromoter-containing plasmids (i.e. NFAT-CD8, B-CD8, cx12lacZ-Oct-1,AP1-LUCIF3H, or cx15IL2) (described below) cut with PvuI and HindIII.NFAT-CD8 contains 3 copies of the NFAT-binding site (-286 to -257; Genesand Dev. (1990) 4, 1823) and cx121acZ-Oct contains 4 copies of theOAP/Oct-1/(ARRE-1) binding site (MCB, (1988) 8, 1715) from the humanIL-2 enhancer; B-CD8 contains 3 copies of the NF B binding site from themurine light chain (EMBO (1990) 9, 4425) and AP1-LUCIF3H contains 5copies of the AP-1 site (5'-TGA-CTCAGCGC-3' SEQ ID NO:3!) from themetallothionen promoter.

In each transfection, 5 μg of expression vector, pCDL-SR (MCB 8, 466-72)(Tac-IL2 receptor-chain), encoding the chimeric receptor TAC/TAC/Z (TTZ)(PNAS 88, 8905-8909), was co-transfected along with various secretedalkaline phosphatase-based reporter plasmids (see map of pSXNeo/IL2 inFIG. 1) in TAg Jurkat cells (a derivative of the human T-cell leukemialine Jurkat stably transfected with the SV40 large T antigen (Northrup,et al., J. Biol. Chem. 1993!). Each reporter plasmid contains amultimerized oligonucleotide of the binding site for a distinct IL-2enhancer-binding transcription factor within the context of the minimalIL-2 promoter or, alternatively, the intact IL-2 enhancer/promoterupstream of the reporter gene. After 24 hours, aliquots of cells(approximately 10⁵) were placed in microtiter wells containing logdilutions of bound anti-TAC (CD25) mAb (33B3.1; AMAC, Westbrook, Me.).As a positive control and to control for transfection efficiency,ionomycin (1 μm) and PMA (25 ng/ml) were added to aliquots from eachtransfection. After an additional 14 hour incubation, the supernatantswere assayed for the alkaline phosphatase activity and these activitieswere expressed relative to that of the positive control samples. Theaddition of I ng/ml FK506 dropped all activity due to NFAT to backgroundlevels, demonstrating that deactivations are in the same pathway as thatblocked by FK506. Each data point obtained was the average of twosamples and the experiment was performed several times with similarresults. See FIG. 5. The data show that with a known extracellularreceptor, one obtains an appropriate response with a reporter gene anddifferent enhancers. Similar results were obtained when a MAb againstthe TcR complex (i.e. OKT3) was employed.

Example 2

Inhibitory Activity of the Immunosuppressant Drugs FK506 and CyclosporinA (CsA) or the Dimeric Derivative Compounds FK1012A (8), FK1012B (5),and CsA dimer (PB-1-218)

Ionomycin (1 μm) and PMA (25 ng/ml) were added to 10⁵ TAg-Jurkat cells.In addition, titrations of the various drugs were added. After 5 hoursthe cells were lysed in mild detergent (i.e. Triton X-100) and theextracts were incubated with the β-galactosidase substrate, MUG (methylgalactosidyl umbelliferone) for 1 hour. A glycine/EDTA stop buffer wasadded and the extracts assayed for fluorescence. Each data pointobtained was the average of two samples and the experiment was performedseveral times with similar results. Curiously, FK1012B appears toaugment mitogen activity slightly at the highest concentration (i.e. 5μg/ml); however, a control experiment shows that FK1012B is notstimulatory by itself. See FIG. 6.

Example 3

Activity of the Dimeric FK506 Derivative, FK1012A, on the ChimericFKBP12/CD3 (1FK3) Receptor

5 μg of the eukaryotic expression vector, pBJ5, (based on pCDL-SR with apolylinker inserted between the 16S splice site and the poly A site),containing the chimeric receptor (1FK3), was co-transfected with 4 μg ofthe NFAT-inducible secreted alkaline phosphatase reporter plasmid,NFAT-SX. As a control, 5 μg of pBJ5 was used, instead of 1FK3/pBJ5, in aparallel transfection. After 24 hours, aliquots of each transfectioncontaining approximately 10⁵ cells were incubated with log dilutions ofthe drug, FK1012A, as indicated. As a positive control and to controlfor transfection efficiency, ionomycin (1 μm) and PMA (25 ng/ml) wereadded to aliquots from each transfection. After an additional 14 hourincubation, the supernatants were assayed for alkaline phosphataseactivity and these activities were expressed relative to that of thepositive control samples. The addition of 2 ng/ml FK506 dropped allstimulations to background levels, demonstrating that the activationsare in the same pathway as that blocked by FK506. Hence, FK506 orcyclosporin will serve as effective antidotes to the use of thesecompounds. Each data point obtained was the average of two samples andthe experiment was performed several times with similar results. SeeFIG. 7.

Example 4A

Activity of the Dimeric FK506 Derivative, FK1012B, on the MyristoylatedChimeric CD3 /FKBP12 (MZF3E) Receptor

We have successfully demonstrated a number of approaches to liganddesign and syntheses, including positive results with FK506-based HODreagents named "FK1012"s. We have found that FK1012s achieve highaffinity, 2:1 binding stoichiometry (K_(d) (1)=0.1 nM; K_(d) (2)=0.8 nM)and do not inhibit calcineurin-mediated TCR signaling. The ligands areneither "immunosuppressive" nor toxic (up to 0.1 mM in cell culture).Similarly, we have prepared a cyclosporin A-based homodimerizing agent,"(CsA)2" which binds to the CsA receptor, cyclophylin, with 1:2stoichiometry, but which does not bind to calcineurin. Thus, likeFK1012s, (CsA)2 does not inhibit signalling pathways and is thus neitherimmunosuppressive nor toxic.

These and other of our examples of ligand-mediated protein associationresulted in the control of a signal transduction pathway. In anillustrative case, this was accomplished by creating an intracellularreceptor comprised of a small fragment of Src sufficient forposttranslational myristoylation (M), the cytoplasmic tail of zeta (Z; acomponent of the B cell receptor was also used), three consecutiveFKBP12s (F3) and a flu epitope tag (E). Upon expressing the constructMZF3E (FIG. 18) in human (Jurkat) T cells, we confirmed that the encodedchimeric protein underwent FK1012-mediated oligomerization. Theattendant aggregation of the zeta chains led to signaling via theendogenous TCR-signaling pathway (FIG. 15), as evidenced by secretion ofalkaline phosphatase (SEAP) in response to an FK1012 (EC₅₀ =50 nM). Thepromoter of the SEAP reporter gene was constructed to betranscriptionally activated by nuclear factor of activated T cells(NFAT), which is assembled in the nucleus following TCR-signaling.FK1012-induced signaling can be terminated by a deaggregation processinduced by a nontoxic, monomeric version of the ligand called FK506-M.

Specifically, 5 μg of the eukaryotic expression vector, pBJ5, containinga myristoylated chimeric receptor was co-transfected with 4 μg NFAT-SX.MZE, MZF1E, MZF2E and MZF3E contain 0, 1, 2, or 3 copies of FKBP12,respectively, downstream of a myristoylated CD3 cytoplasmic domain (seeFIG. 2). As a control, 5 μg of pBJ5 was used in a parallel transfection.After 24 hours, aliquots of each transfection containing approximately10⁵ cells were incubated with log dilutions of the drug, FK1O12B, asindicated. As a positive control and to control for transfectionefficiency, ionomycin (1 μm) and PMA (25 ng/ml) were added to aliquotsfrom each transfection. After an additional 12 hour incubation, thesupernatants were assayed for alkaline phosphatase activity and theseactivities were expressed relative to that of the positive controlsamples. The addition of 1 ng/ml FK506 dropped all stimulations to nearbackground levels, demonstrating that the activations are in the samepathway as that blocked by FK506. This result is further evidence of thereversibility of the subject cell activation. Each data point obtainedwas the average of two samples and the experiment was performed severaltimes with similar results. See FIG. 8. The myristoylated derivativesrespond to lower concentrations of the ligand by about an order ofmagnitude and activate NF-AT dependent transcription to comparablelevels, but it should be noted that the ligands are different. CompareFIGS. 7 and 8.

In vivo FK1012-Induced Protein Dimerization

We next wanted to confirm that intracellular aggregation of the MZF3Ereceptor is indeed induced by the FK1012. The influenza haemagglutininepitope-tag (flu) of the MZF3E-construct was therefore exchanged with adifferent epitope-tag (flag-M2). The closely related chimeras,MZF3E_(flu) and MZF3E_(flag), were coexpressed in Jurkat T cells.Immunoprecipitation experiments using anti-Flag-antibodies coupled toagarose beads were performed after the cells were treated with FK1012A.In the presence of FK1012A (1 μM) the protein chimera MZF3E_(flag)interacts with MZF3E_(flu) and is coimmunoprecipitated withMZF3E_(flag). In absence of FK1012A, no coimmunoprecipitation ofMZF3E_(flu) is observed. Related experiments with FKBP monomerconstructs MZF1E_(flu) and MZF1E_(flag), which do not signal, revealedthat they are also dimerized by FK1012A (FIG. 19). This reflects therequirement for aggregation observed with both the endogenous T cellreceptor and our artificial receptor MZF3E.

FK1012-Induced Protein-Tyrosine Phosphorylation

The intracellular domains of the TCR, CD3 and zeta-chains interact withcytoplasmic protein tyrosine kinases following antigen stimulation.Specific members of the Src family (lck and/or fyn) phosphorylate one ormore tyrosine residues of activation motifs within these intracellulardomains (tyrosine activation motif, TAM). The tyrosine kinase ZAP-70 isrecruited (via its two SH2 domains) to the tyrosine phosphorylatedT-cell-receptor, activated, and is likely to be involved in the furtherdownstream activation of phospholipase C. Addition of either anti-CD3MAb or FK1012A to Jurkat cells stably transfected with MZF3E resulted inthe recruitment of kinase activity to the zeta-chain as measured by anin vitro kinase assay following immunoprecipitation of the endogenous Tcell receptor zeta chain and the MZF3E-construct, respectively. Tyrosinephosphorylation after treatment of cells with either anti-CD3 MAb orFK1012 was detected using monoclonal alpha-phosphotyrosine antibodies.Whole cell lysates were analysed at varying times after stimulation. Asimilar pattern of tyrosine-phosphorylated proteins was observed afterstimulation with either anti-CD3 MAb or FK1012. The pattern consisted ofa major band of 70 kDa, probably ZAP-70, and minor bands of 120 kDa, 62kDa, 55 kDa and 42 kDa.

Example 4(B)

Regulation of Programmed Cell Death with Immunophilin-Fas AntigenChimeras

The Fas antigen is a member of the nerve growth factor (NGF)/tumornecrosis factor (TNF) receptor superfamily of cell surface receptors.Crosslinking of the Fas antigen with antibodies to its extracellulardomain activates a poorly understood signaling pathway that results inprogrammed cell death or apoptosis. The Fas antigen and its associatedapoptotic signaling pathway are present in most cells including possiblyall tumor cells. The pathway leads to a rapid and unique cell death (2h) that is characterized by condensed cytoplasm, the absence of aninflammatory response and fragmentation of nucleosomal DNA, none ofwhich are seen in necrotic cell death.

We have also developed a second, inducible signaling system that leadsto apoptotic cell death. Like the MZF3E pathway, this one is initiatedby activating an artificial receptor that is the product of aconstitutively expressed "responder" gene. However, the new pathwaydiffers from the first in that our HOD reagents induce the synthesis ofproducts of an endogenous pathway rather than of the product of atransfected, inducible (e.g., reporter) gene.

Gaining control over the Fas pathway could have important implicationsfor biological research and medicine in the future. Transgenic animalsmight be created with "death" responder genes under the control ofcell-specific promoters. Target cells could then be chemically ablatedin the adult animal by treating it with a HOD reagent. In this way, therole of specific brain cells in memory or cognition or immune cells inthe induction and maintenance of autoimmune disorders could be assessed.Death responder genes might be introduced into tumors using the humangene therapy technique developed by M. Blaese and co-workers (Culver etal, Science 256 5063 (1992): 1550-2) and then subsequently activated bytreating the patient with a HOD reagent (in analogy to the "gancyclovir"gene therapy clinical trials recently reported for the treatment ofbrain tumors). Finally, we contemplate a component of gene therapy inthe future that would involve the coadministration of a death-respondergene together with the therapeutic gene. This would provide a "failsafe"component to gene therapy. If something were to go awry (a commonlydiscussed concern is an integration-induced loss of a tumor suppressorgene leading to cancer), the gene therapy patient could take a"failsafe" pill that would kill all transfected cells. This conceptcaused us to focus on the development of an orthogonal system of HODreagents. Thus, we desired a second set of reagents that have nopossibility of cross-reacting with the first, which would be used toturn on or off the transcription of therapeutic genes.

A chimeric cDNA has been constructed consisting of three FKBP12 domainsfused to the cytoplasmic signaling domain of the Fas antigen (FIG. 20).This construct, when expressed in human Jurkat and murine D10 T cells,can be induced to dimerize by an FK1012 reagent and initiate a signalingcascade resulting in FK1012-dependent apoptosis. The LD₅₀ forFK1012A-mediated death of cells transiently transfected with MFF3E is 15nM as determined by a loss of reporter gene activity (FIG. 20; for adiscussion of the assay, see legend to FIG. 21). These data coincidewith measurements of cell death in stably transfected cell lines. Sincethe stable transfectants represent a homogeneous population of cells,they have been used to ascertain that death is due to apoptosis ratherthan necrosis (membrane blebbing, nucleosomal DNA fragmentation).However, the transient transfection protocol requires much less work andhas therefore been used as an initial assay system, as described below.

Example 4(C)

Regulation of Programmed Cell Death with Cyclophilin-Fas AntigenChimeras

We have also prepared a series of cyclophilin C-Fas antigen constructsand assayed their ability to induce (CsA)2-dependent apoptosis intransient expression assays (FIG. 21A). In addition, (CsA)2-dependentapoptosis has been demonstrated with human Jurkat T cells stablytransfected with the most active construct in the series, MC3FE(M=myristoylation domain of Src, C=cyclophilin domain, F=cytoplasmictail of Fas, E=flu epitope tag). The cytoplasmic tail of Fas was fusedeither before or after 1, 2, 3, or 4 consecutive cyclophilin domains.Two control constructs were also prepared that lack the Fas domain. Inthis case we observed that the signaling domain functions only whenplaced after the dimerization domains. (The zeta chain constructs signalwhen placed either before or after the dimerization domains.) Both theexpression levels of the eight signaling constructs, as ascertained byWestern blotting, and their activities differed quantitatively (FIG.21B). The optimal system has thus far proved to be MC3FE. The LD50 for(CsA)2-mediated cell death with MC3FE is ˜200 nM. These data demonstratethe utility of the cyclophilin-cyclosporin interactions for regulatingintracellular protein association and illustrate an orthogonal reagentsystem that will not cross-react with the FKBP12-FK1012 system. Further,in this case, the data show that only dimerization and not aggregationis required for initiation of signal transduction by the Fas cytoplasmictail.

Mutation of the N-terminal glycine of the myristoylation signal to analanine prevents myristoylation and hence membrane localization. We havealso observed that the mutated construct (AMFF3E) was equally potent asan inducer of FK1012-dependent apoptosis, indicating that membranelocalization is not necessary for Fas-mediated cell death.

Example 5

Construction of Murine Signalling Chimeric Protein

The various fragments were obtained by using primers described in FIG. 4SEQ ID NOS:4, 6, 8, 10, 12, 14, 16, 17, 23 and 35!. In referring toprimer numbers, reference should be made to FIG. 4.

An approximately 1.2 kb cDNA fragment comprising the I-E chain of themurine class II MHC receptor (Cell, 32, 745) was used as a source of thesignal peptide, employing P#6048 SEQ ID NO; 4! and P#6049 SEQ ID NO: 6!to give a 70 bp SacII-XhoI fragment using PCR as described by thesupplier (Promega). A second fragment was obtained using a plasmidcomprising Tac (IL2 receptor chain) joined to the transmembrane andcytoplasmic domains of CD3 (PNAS, 88, 8905). Using P#6050 SEQ ID NO:8!and P#6051, SEQ ID NO:10!, a 320 bp XhoI-EcoRI fragment was obtained byPCR comprising the transmembrane and cytoplasmic domains of CD3. Thesetwo fragments were ligated and inserted into a SacII-EcoRI digestedpBluescript (Stratagene) to provide plasmid, SPZ/KS.

To obtain the binding domain for FK506, plasmid rhFKBP (provided by S.Schreiber, Nature (1990) 346, 674) was used with P#6052 SEQ ID NO:33!and P#6053 SEQ ID NO:35! to obtain a 340 bp XhoI-SalI fragmentcontaining human FKBP12. This fragment was inserted into pbluescriptdigested with XhoI and SalI to provide plasmid FK12/KS, which was thesource for the FKBP12 binding domain. SPZ/KS was digested with XhoI,phosphatased (cell intestinal alkaline phosphatase; CIP) to preventself-annealing, and combined with a 10-fold molar excess of theXhoI-SalI FKBP12-containing fragment from FK12/KS. Clones were isolatedthat contained monomers, dimers, and trimers of FKBP12 in the correctorientation. The clones 1FK1/KS, 1FK2/KS, and 1FK3/KS are comprised ofin the direction of transcription; the signal peptide from the murineMHC class II gene I-E, a monomer, dimer or trimer, respectively, ofhuman FKBP12, and the transmembrane and cytoplasmic portions of CD3.Lastly, the SacII-EcoRI fragments were excised from pBluescript usingrestriction enzymes and ligated into the polylinker of pBJ5 digestedwith SacII and EcoRI to create plasmids 1FK1/pBJ5, 1FK2/pBJ5, and1FK3/pBJ5, respectively. See FIGS. 3 and 4.

Example 6

A. Construction of Intracellular Signaling Chimera

A myristoylation sequence from c-src was obtained from Pellman, et al.,Nature 314, 374, and joined to a complementary sequence of CD3 toprovide a primer which was complementary to a sequence 3' of thetransmembrane domain, namely P#8908 SEQ ID NO:23!. This primer has aSacII site adjacent to the 5' terminus and a XhoI sequence adjacent tothe 3' terminus of the myristoylation sequence. The other primer P#8462SEQ ID NO:12! has a SalI recognition site 3' of the sequencecomplementary to the 3' terminus of CD3, a stop codon and an EcoRIrecognition site. Using PCR, a 450 bp SacII-EcoRI fragment was obtained,which was comprised of the myristoylation sequence and the CD3 sequencefused in the 5' to 3' direction. This fragment was ligated intoSaclI/EcoRI-digested pBJ5(XhoI)(SalI) and cloned, resulting in plasmidMZ/pBJ5. Lastly, MZ/pBJ5 was digested with SalI, phosphatased, andcombined with a 10-fold molar excess of the XhoI-SalI FKBP12-containingfragment from FK12/KS and ligated. After cloning, the plasmidscomprising the desired constructs having the myristoylation sequence,CD3 and FKBP12 multimers in the 5'-3' direction were isolated andverified as having the correct structure. See FIGS. 2 and 4.

B. Construction of Expression Cassettes for Intracellular SignalingChimeras

The construct MZ/pBJ5 (MZE/pBJ5) is digested with restriction enzymesXhoI and SalI, the TCR ζ fragment is removed and the resulting vector isligated with a 10 fold excess of a monomer, dimer, trimer or higherorder multimer of FKBP12 to make MF1E, MF2E, MF3E or MF_(n) E/pBJ5.Active domains designed to contain compatible flanking restriction sites(i.e. XhoI and SalI) can then be cloned into the unique XhoI or SalIrestriction sites of MF_(n) E/pBJ5.

Example 7

Construction of Nuclear Chimera

A. GAL4 DNA Binding Domain--FKBP Domain(s)--Epitope Tag

The GAL4 DNA binding domain (amino acids 1-147) was amplified by PCRusing a 5' primer (#37) that contains a SacII site upstream of a Kozaksequence and a translational start site, and a 3' primer (#38) thatcontains a SalI site. The PCR product was isolated, digested with SacIIand SaII, and ligated into pBluescript II KS (+) at the SacII and SaIISites, generating the construct pBS-GAL4. The construct was verified bysequencing. The SacII/SaII fragment from pBS-GAL4 was isolated andligated into the IFK1/pBJ5 and IFK3/pBJ5 constructs (containing themyristoylation sequence, see Example 6) at the SacII and Xhol sites,generating constructs GF1E, GF2E and GF3E.

5' end of PCR amplified product: ##STR1##

3' end of PCR amplified product: ##STR2##

B. HNF1 Dimerization/DNA Binding Domain--FKBP domain(s)--Tag

The HNF1a dimerization/DNA binding domain (amino acids 1-282) wasamplified by PCR using a 5' primer (#39) that contains a SacII siteupstream of a Kozak sequence and a translational start site, and a 3'primer (#40) that contains a SalI site. The PCR product was isolated,digested with SacII and SalI, and ligated into pBluescript II KS (+) atthe SacII and SalI sites, generating the construct pBS-HNF. Theconstruct was verified by sequencing. The SacII/SalI fragment frompBS-HNF was isolated and ligated into the IFK1/pBJ5 and IFK3/pBJ5constructs at the SacII and XhoI sites, generating constructs HF1E, HF2Eand HF3E.

5' End of PCR Amplified Product ##STR3##

3' end of PCR amplified product: ##STR4##

C. FKBP Domain(s)--VP16 Transcrip. Activation Domain(s)--Epitope Tag

These constructs were made in three steps: (i) a construct was createdfrom IFK3/pBJ5 in which the myristoylation sequence was replaced by astart site immediately upstream of an XhoI site, generating constructSF3E; (ii) a nuclear localization sequence was inserted into the XhoIsite, generating construct NF3E; (iii) the VP16 activation domain wascloned into the SalI site of NF3E, generating construct NF3V1E.

(i). Complementary oligonucleotides (#45 and #46) encoding a Kozaksequence and start site flanked by SacII and XhoI sites were annealed,phosphorylated and ligated into the SacII and XhoI site of MF3E,generating construct SF3E.

Insertion of generic start site ##STR5##

(ii). Complementary oligonucleotides (#47 and #48) encoding the SV40 Tantigen nuclear localization sequence flanked by a 5' SalI site and a 3'XhoI site were annealed, phosphorylated and ligated into the Xhol siteof SF1E, generating the construct NF1E. The construct was verified byDNA sequencing. A construct containing the mutant or defective form ofthe nuclear localization sequence, in which a threonine is substitutedfor the lysine at position 128, was also isolated. This is designatedNF1E-M. Multimers of the FKBP12 domain were obtained by isolating theFKBP12 sequence as an Xhol/SalI fragment from pBS-FKBP12 and ligatingthis fragment into NF1LE linearized with Xhol. This resulted in thegeneration of the constructs NF2E and NF3E.

Insertion of NLS into generic start site ##STR6##

Threonine at position 128 results in a defective NLS.

(iii). The VP16 transcriptional activation domain (amino acids 413-490)was amplified by PCR using a 5' primer (#43) that contains SalI site anda 3' primer (#44) that contains an XhoI site. The PCR product wasisolated, digested with SalI and XhoI, and ligated into MF3E at the XhoIand SalI sites, generating the construct MV1E. The construct wasverified by sequencing. Multimerized VP16 domains were created byisolating the single VP16 sequence as a XhoI/SalI fragment from MV1E andligating this fragment into MV1E linearized with XhoI. Constructs MV2E,MV3E and MV4E were generated in this manner. DNA fragments encoding oneor more multiple VP16 domains were isolated as Xhol/SalI fragments fromMV1E or MV2E and ligated into NF1E linearized with SalI, generating theconstructs NF1VLE and NF1V3E. Multimers of the FKBP12 domain wereobtained by isolating the FKBP12 sequence as an XhoI/SalI fragment frompBS-FKBP12 and ligating this fragment into NF1VlE linearized with XhoI.This resulted in the generation of the constructs NF2V1E and NF3V1E.

5' end of PCR amplified product: ##STR7##

3' end of PCR amplified product: ##STR8## Oligonucleotides #37 38mer/0.2 um/OFF 5'CGACACCGCGGCCACCATGAAGCTACTGTCTT CTATCG SEQ ID NO:41!

#38 28 mer/0.2 um/OFF 5'CGACAGTCGACCGATACAGTCAACTGTC SEQ ID NO:42!

#39 34 mer/0.2 um/OFF 5'CGACACCGCGGCCACCATGGTTTCTAAGCTGAGC SEQ ID NO:49!

#40 28 mer/0.2 um/OFF 5'CGACAGTCGACCAACTTGTGCCGGAAGG SEQ ID NO:48!

#43 29 mer/0.2 um/OFF 5'CGACAGTCGACGCCCCCCCGACCGATGTC SEQ ID NO:61!

#44 26 mer/0.2 um/OFF 5'CGACACTCGAGCCCACCGTACTCGTC SEQ ID NO:62!

#45 26 mer/0.2 um/OFF 5'GGCCACCATGC SEQ ID NO:53 !

#46 18 mer/0.2 um/OFF 5'TCGAGCATGGTGGCCGC SEQ ID NO:55!

#47 27 mer/0.2 um/OFF 5'TCGACCCTAAGA-(C/A)-GAAGAGAAAGGTAC SEQ ID NO:56!

#48 27 mer/0.2 um/OFF 5'TCGAGTACCTTTCTCTTC-(G/T)-TCTTAGGG SEQ ID NO:57!

Example 8

Demonstration of Transcriptional Induction

Jurkat TAg cells were transfected with the indicated constructs (5 μg ofeach construct) by electroporation (960 μF, 250 v). After 24 hours, thecells were resuspended in fresh media and aliquoted. Half of eachtransfection was incubated with the dimeric FK506 derivative, (Example14) at a final concentration of 1 μM. After 12 hours, the cells werewashed and cellular extracts were prepared by repeated freeze-thaw.Chloramphenicol acetyltransferase (CAT) activity was measured bystandard protocols. Molecular Cloning: A Laboratory Manual, Sambrook etal. eds. (1989) CSH Laboratory, pp. 16-59 ff. The data (FIG. 22)demonstrates CAT activity present in 70 μL of extract (total extractvolume was 120 μL) after incubation at 37° C. for 18 hours. The samplesemployed in the assays are as follows:

1. G5E4TCAT (GAL4-CAT reporter plasmid)

2. G5E4TCAT, GAL4-VP16

3. G5E4TCAT, NF3V1E

4. G5E4TCAT, GF2E

5. G5E4TCAT, GF2E, NF3V1E

6. G5E4TCAT, GF3E, NF3V1E

Synthetic Chemistry Examples

As indicated elsewhere, compounds of particular interest at present asoligomerization agents have the following structure:

    linker--{rbm.sub.1,rbm.sub.2, . . . rbm.sub.n }.

wherein "linker" is a linker moiety such as described herein which iscovalently linked to "n" (an integer from 2 to about 5, ususally 2 or 3)receptor binding moieties ("rbm"'s) which may be the same or different.As discussed elsewhere herein, the receptor binding moiety is a ligand(or analog thereof) for a known receptor, such as are enumerated inSection V(C), and including FK506, FK520, rapamycin and analogs thereofwhich are capable of binding to an FKBP; as well as cyclosporins,tetracyclines, other antibiotics and macrolides and steroids which arecapable of binding to respective receptors.

The linker is a bi- or multi-functional molecule capable of beingcovalently linked ("--") to two or more receptor binding moieties.Illustrative linker moieties are disclosed in Section VI(A) and in thevarious Examples and include among others C2-C20 alkylene, C4-C18azalkylene, C6-C24 N-alkylene azalkylene, C6-C18 arylene, C8-C24ardialkylene and C8-C36 bis-carboxamido alkylene.

These compounds may be prepared using commercially available materialsand/or procedures known in the art. Engineered receptors for thesecompounds may be obtained as described infra. Compounds of particularinterest are those which bind to a receptor with a Kd of less than 10⁻⁶,preferably less than about 10⁻⁷ and even more preferably, less than10⁻⁸.

One subclass of oligomerizing agents of interest are those in which oneor more of the receptor binding moieties is FK506, an FK-506-typecompound or a derivative thereof, wherein the receptor binding moietiesare covalently attached to the linker moiety through the allyl group atC21 (using FK506 numbering) as per compound 5 or 13 in FIG. 23A, orthrough the cyclohexyl ring (C29-C34), e.g. through the C32 hydroxyl asper compounds 8, 16, 17 in FIG. 23B. Compounds of this class may beprepared by adaptation of methods disclosed herein, including in theexamples which follow.

Another subclass of oligomerizing agents of interest are those in whichat least one of the receptor binding moieties is FK520 or a derivativethereof, wherein the molecules of FK520 or derivatives thereof arecovalently attached to the linker moiety as in FK1040A or FK 1040B inFIG. 10. Compounds of this class may be prepared by adaptation of Scheme1 in FIG. 10, Scheme 2 in FIGS. 11A and 11B or Scheme 3 in FIG. 12 andFIG. 13.

A further subclass of oligomerizing agents of interest are those inwhich at least one of the receptor binding moieties is cyclosporin A ora derivative.

It should be appreciated that these and other oligomerizing agents ofthis invention may be homo-oligomerizing reagents (where the rbm's arethe same) or hetero-oligomerizing agents (where the rbm's aredifferent). Hetero-oligomerizing agents may be prepard by analogy to theprocedures presented herein, including Scheme 3 in FIG. 13 and asdiscussed elsewhere herein.

The following synthetic examples are intended to be illustrative.

A. General Procedures

All reactions were performed in oven-dried glassware under a positivepressure of nitrogen or argon. Air and moisture sensitive compounds wereintroduced via syringe or cannula through a rubber septum.

B. Physical Data

Proton magnetic resonance spectra (¹ H NMR) were recorded on BrukerAM-500 (500 MHz), and AM-400 (400 MHz) spectrometers. Chemical shiftsare reported in ppm from tetramethylsilane using the solvent resonanceas an internal standard (chloroform, 7.27 ppm). Data are reported asfollows: chemical shift, multiplicity (s=singlet, d=doublet, t=triplet,q=quartet, br=broadened, m=multiplet), coupling constants (Hz),integration. Low and high-resolution mass spectra were obtained.

C. Chromatography

Reactions were monitored by thin layer chromatography (TLC) using E.Merck silica gel 60F glass plates (0.25 mm). Components were visualizedby illumination with long wave ultraviolet light, exposed to iodinevapor, and/or by dipping in an aqueous ceric ammonium molybdate solutionfollowed by heating. Solvents for chromatography were HPLC grade. Liquidchromatography was performed using forced flow (flash chromatography) ofthe indicated solvent system on E. Merck silica gel 60 (230-400 mesh).

D. Solvents and Reagents

All reagents and solvents were analytical grade and were used asreceived with the following exceptions. Tetrahydrofuran (THF), benzene,toluene, and diethyl ether were distilled from sodium metal benzophenoneketyl. Triethylamine and acetonitrile were distilled from calciumhydride. Dichloromethane was distilled from phosphorous pentoxide.Dimethylformamide (DMF) was distilled from calcium hydride at reducedpressure and stored over 4 Å molecular sieves.

Preparation of FK506 Derivatives

Example 9

Hydroboration/Oxidation of FKSO6-TBS₂ (1 to 2)

The hydroboration was performed according to the procedure of Evans(Evans, et al., JACS (1992) 114, 6679; ibid. (1992) 6679-6685). (SeeHarding, et al., Nature (1989) 341, 758 for numbering.) A 10-mL flaskwas charged with 24,32-bis (tert-butyldimethylsilyl)oxy!-FK506 (33.8 mg,0.033 mmol) and Rh(nbd)(diphos-4)!BF₄ (3.1 mg, 0.004 mmol, 13 mol %).The orange mixture was dissolved in toluene (2.0 mL) and the solvent wasremoved under reduced pressure over four hours. The flask was carefullypurged with nitrogen and the orangish oil was dissolved in THF (3.0 mL,10 mM final concentration) and cooled to 0° C. with an ice water bath.Catecholborane (98 μL, 0.098 mmol, 1.0M solution in THF, 3.0 equiv.) wasadded via syringe and the resulting solution was stirred at 0° C. for 45min. The reaction was quenched at 0° C. with 0.2 mL of THF/EtOH (1:1)followed by 0.2 mL of pH 7.0 buffer (Fisher; 0.05M phosphate) then 0.2mL of 30% H₂ O₂. The solution was stirred at room temperature for atleast 12 h. The solvent was removed under reduced pressure and theremaining oil was dissolved in benzene (10 mL) and washed with saturatedaqueous sodium bicarbonate solution (10 mL). The phases were separatedand the aqueous phase was back-extracted with benzene (2×10 mL). Theorganic phases were combined and washed once with saturated aqueoussodium bicarbonate solution (10 mL). The benzene phase was dried withMgSO₄, concentrated, and subjected to flash chromatography (2:1hexane:ethyl acetate) providing the desired primary alcohol as a clear,colorless oil (12.8 mg, 0.012 mmol, 37%).

Preparation of Mixed Carbonate (2 to 3)

The preparation of the mixed carbonate was accomplished by the method ofGhosh (Ghosh, et al., Tetrahedron Lett. (1992) 33, 2781-2784). A 10-mLflask was charged with the primary alcohol (29.2 mg, 0.0278 mmol) andbenzene (4 mL). The solvent was removed under reduced pressure over 60min. The oil was dissolved in acetonitrile (2.0 mL, 14 mM finalconcentration) and stirred at 20° C. as triethylamine (77 μL, 0.56 mmol)was added. N,N'-disuccinimidyl carbonate (36 mg, 0.14 mmol) was added inone portion and the solution was stirred at 20° C. for 46 h. Thereaction mixture was diluted with dichloromethane and washed withsaturated aqueous sodium bicarbonate solution (10 mL). The phases wereseparated and the aqueous layer was back-extracted with dichloromethane(2×10 mL). The organic phases were combined and dried (MgSO₄),concentrated, and subjected to flash chromatography (3:1 to 2:1 to 1:1hexane:ethyl acetate). The desired mixed carbonate was isolated as aclear, colorless oil (16.8 mg, 0.014 mmol, 51%).

Dimerization of FK506 (3 to 4)

A dry, 1-mL conical glass vial (Kontes Scientific Glassware) was chargedwith the mixed carbonate (7.3 mg, 0.0061 mmol) and acetonitrile (250 μL,25 mM final concentration). Triethylamine (10 μL, 0.075 mmol) was addedfollowed by p-xylylenediamine (8.3 μL, 0.0027 mmol, 0.32M solution inDMF). The reaction stirred 22 h at 20° C. and was quenched by dilutionwith dichloromethane (10 mL). The solution was washed with saturatedaqueous sodium bicarbonate solution (10 mL). The phases were separatedand the aqueous layer was back-extracted with dichloromethane (2×10 mL).The organic phases were combined and dried (MgSO₄), concentrated, andsubjected to flash chromatography (3:1 to 2:1 to 1:1 hexane:ethylacetate) providing the desired protected dimer as a clear, colorless oil(4.3 mg, 1.9 μmol, 70%).

Deprotection of the FK506 Dimer (4 to 5)

The protected dimer (3.3 mg, 1.4 μmol) was placed in a 1.5-mLpolypropylene tube fitted with a spin vane. Acetonitrile (0.5 mL, 3 mMfinal concentration) was added and the solution stirred at 20° C. as HF(55 μL, 48% aqueous solution; Fisher) was added. The solution wasstirred 18 h at room temperature. The deprotected FK506 derivative wasthen partitioned between dichloromethane and saturated aqueous sodiumbicarbonate in a 15-mL test tube. The tube was vortexed extensively tomix the phases and, after separation, the organic phase was removed witha pipet. The aqueous phase was back-extracted with dichloromethane (4×2mL), and the combined organic phases were dried (MgSO₄), concentratedand subjected to flash chromatography (1:1:1 hexane:THF:ether to 1:1THF:ether) providing the desired dimer as a clear, colorless oil (1.7mg, 0.93 μmol, 65%).

Following the above procedure, other monoamines and diamines may beused, such as benzylamine (14) octamethylenediamine,decamethylenediamine, etc.

Example 10

Reduction of FK506 with L-Selectride (FK506 to 6)

Danishefsky and coworkers have shown that the treatment of FK506 withL-Selectride provides 22-dihydro-FK506 with a boronate ester engagingthe C24 and C22 hydroxyl groups (Coleman and Danishefsky, Heterocycles(1989) 28, 157-161; Fisher, et al., J. Org. Chem. (1991) 56, 2900-2907).

Preparation of the Mixed Carbonate (6 to 7)

A 10-mL flask was charged with 22-dihydro-FK506-sec-butylboronate (125.3mg, 0.144 mmol) and acetonitrile (3.0 mL, 50 mM final concentration) andstirred at room temperature as triethylamine (200 μL, 1.44 mmol, 10equiv.) was added to the clear solution. N,N'-disuccinimidyl carbonate(184.0 mg, 0.719 mmol) was added in one portion, and the clear solutionwas stirred at room temperature for 44 h. The solution was diluted withethyl acetate (20 mL) and washed with saturated aqueous sodiumbicarbonate (10 mL) and the phases were separated. The aqueous phase wasthen back-extracted with ethyl acetate (2×10 mL), and the organic phaseswere combined, dried (MgSO₄), and the resulting oil was subjected toflash chromatography (1:1 to 1:2 hexane:ethyl acetate) providing thedesired mixed carbonate as a clear, colorless oil (89.0 mg, 0.088 mmol,61%).

Dimerization of FK506 Mixed Carbonate (7 to 8)

A dry, 1-mL conical glass vial (Kontes Scientific Glassware) was chargedwith the mixed carbonate (15.0 mg, 0.0148 mmol) and dichloromethane (500μL, 30 mM final concentration). The solution was stirred at roomtemperature as triethylamine (9 μL, 0.067 mmol, 10 equiv.) was addedfollowed by p-xylylenediamine (0.8 mg, 0.0059 mmol). The reactionstirred 16 h at 20° C. and was quenched by dilution with dichloromethane(5 mL). The solution was washed with saturated aqueous sodiumbicarbonate solution (5 mL). The phases were separated and the aqueouslayer was back-extracted with dichloromethane (2×5 mL). The organicphases were combined and dried (MgSO₄), concentration, and subjected toflash chromatography (1:1 to 1:2 hexane:ethyl acetate) providing thedesired dimer as a clear, colorless oil (7.4 mg, 3.8 μmol, 65%).

Following the above procedure, other, monoamines, diamines or triaminesmay be used in place of the xylylenediamine, such as benzylamine (15),octylenediamine, decamethylenediamine (16), bis-p-dibenzylamine,N-methyl diethyleneamine, tris-aminoethylamine (17),tris-aminopropylamine, 1,3,5-triaminomethylcyclohexane, etc.

Example 11

Oxidative Cleavage and Reduction of FK506 (1 to 9)

The osmylation was performed according to the procedure of Kelly(VanRheenen, et al., Tetrahedron Lett. (1976) 17, 1973-1976). Thecleavage was performed according to the procedure of Danishefsky (Zell,et al., J. Org. Chem. (1986) 51, 5032-5036). The aldehyde reduction wasperformed according to the procedure of Krishnamurthy (J. Org. Chem.,(1981) 46, 4628-4691). A 10 mL flask was charged with 24,32-bistert-butyldimethylsilyl)oxy!-FK506 (84.4 mg, 0.082 mmol),4-methylmorpholine N-oxide (48 mg, 0.41 mmol, 5 equiv), and THF (2.0 mL,41 mM final concentration). Osmium tetroxide (45 μL, 0.008 mmol, 0.1equiv) was added via syringe. The clear, colorless solution was stirredat room temperature for 5 hr. The reaction was then diluted with 50%aqueous methanol (1.0 mL) and sodium periodate (175 mg, 0.82 mmol, 10equiv) was added in one portion. The cloudy mixture was stirred 40 minat room temperature, diluted with ether (10 mL), and washed withsaturated aqueous sodium bicarbonate solution (5 mL). The phases wereseparated and the aqueous layer was back-extracted with ether (2×5 mL).The combined organic layers were dried (MgSO₄) and treated with solidsodium sulfite (50 mg). The organic phase was then filtered andconcentrated and the oil was subjected to flash chromatography (3:1 to2:1 hexane:ethyl acetate) providing the intermediate, unstable aldehyde(53.6 mg) as a clear, colorless oil. The aldehyde was immediatelydissolved in THF (4.0 mL) and cooled to -78° C. under an atmosphere ofnitrogen, and treated with lithium tris (3-ethyl-3-pentyl)oxy!aluminumhydride (0.60 mL, 0.082 mmol, 0.14M solution in THF, 1.0 equiv). Theclear solution was allowed to stir for 10 min at -78° C. then quenchedby dilution with ether (4 mL) and addition of saturated aqueous ammoniumchloride (0.3 mL). The mixture was allowed to warm to room temperatureand solid sodium sulfate was added to dry the solution. The mixture wasthen filtered and concentrated and the resulting oil was subjected toflash chromatography (2:1 hexane:ethyl acetate) giving the desiredalcohol as a clear, colorless oil (39.5 mg, 0.038 mmol, 47%).

Preparation of Mixed Carbonate (9 to 10)

The preparation of the mixed carbonate was accomplished by the method ofGhosh, et al., Tetrahedron Lett. (1992) 33, 2781-2784). A 10 mL flaskwas charged with the primary alcohol (38.2 mg, 0.0369 mmol) andacetonitrile (2.0 mL, 10 mM final concentration) and stirred at roomtemperature as 2,6-lutidine (43 μL, 0.37 mmol, 10 equiv) was added.N,N'-disuccinimidyl carbonate (48 mg, 0.18 mmol) was added in oneportion and the solution was stirred at room temperature for 24 h. Thereaction mixture was diluted with ether (10 mL) and washed withsaturated aqueous sodium bicarbonate solution (10 mL). The phases wereseparated and the aqueous layer was back-extracted with ether (2×10 mL).The organic phases were combined and dried (MgSO₄), concentrated, andsubjected to flash chromatography (2:1 to 1:1 hexane:ethyl acetate). Thedesired mixed carbonate was isolated as a clear, colorless oil (32.6 mg,0.028 mmol, 75%).

Preparation of Benzyl Carbamate (10 to 11)

A dry, 1 mL conical glass vial (Kontes Scientific Glassware) was chargedwith the mixed carbonate 10 (8.7 mg, 0.0074 mmol) and acetonitrile (500μL, 15 mM final concentration). The solution was stirred at roomtemperature as triethylamine (10 μL, 0.074 mmol, 10 equiv) was addedfollowed by benzylamine (1.6 μL, 0.015 mmol, 2 equiv). The reactionstirred 4 h at room temperature. The solvent was removed with a streamof dry nitrogen and the oil was directly subjected to flashchromatography (3:1 to 2:1 hexane:ethyl acetate) providing the desiredprotected monomer as a clear, colorless oil (6.2 mg, 5.3 μmol, 72%).

The protected monomer (0.2 mg, 5.3 μmol) was placed in a 1.5 mLpolypropylene tube fitted with a spin vane. Acetonitrile (0.5 mL, 11 mMfinal concentration) was added and the solution stirred at roomtemperature as HF (55 μL, 48% aqueous solution; Fisher, 3.0N finalconcentration) was added. The solution was stirred 18 h at roomtemperature. The deprotected FK506 derivative was then partitionedbetween dichloromethane and saturated aqueous sodium bicarbonate in a 15mL test tube. The tube was vortexed extensively to mix the phases and,after separation, the organic phase was removed with a pipet. Theaqueous phase was back-extracted with dichloromethane (4×2 mL), and thecombined organic phases were dried (MgSO₄), concentrated and subjectedto flash chromatography (1:1 to 0:1 hexane:ethyl acetate) providing forthe desired deprotected benzylcarbamate as a clear, colorless oil (3.9mg, 4.1 μmol, 78%).

By replacing the benzylamine with a diamine such as xylylenediamine(12), hexamethylenediamine, octamethylenediamine, decamethylenediamine(13) or other diamines, dimeric compounds of the subject invention areprepared.

Example 12

Preparation of the Mixed Carbonate of FK506 (12)

A 10-mL flask was charged with 24, 32-bis(tert-butyldimethylsilyl)oxy!-FK506 (339.5 mg., 0.329 mmol),4-methylmorpholine N-oxide (193 mg, 1.64 mmol, 5 equiv), water (0.20 mL)and THF (8.0 mL, 41 mN final concentration). Osmium tetroxide (0.183 mL,0.033 mmol, 0.1 equiv, 0.18M soln in water) was added via syringe. Theclear, colorless solution was stirred at room temperature for 4.5 h. Thereaction was diluted with 50% aqueous methonol (4.0 mL) and sodiumperiodate (700 mg, 3.29 mmol, 10 equiv) was added in one portion. Thecloudy mixture was stirred 25 min at room temperture, diluted with ether(20 mL), and washed with saturated aqueous sodium bicarbonate solution(10 mL). The phases were separated and the aqueous layer wasback-extracted with ether (2×10 mL). The combined organic layers weredried over MgSO₄ and solid sodium sulfite (50 mg). The organic phase wasthen filtered and concentrated and the resulting aldehyde wasimmediately dissolved in THF (8.0 mL) and cooled to -78 ° C. under anatmosphere of nitrogen, and treated with lithium tris(3-ethyl-3-pentyl)oxy! aluminum hydride (2.35 mL, 0.329 mmol, 0.14Msolution of THF, 1.0 equiv). The clear solution was allowed to stir for60 min at -78 ° C. (monitored closely by TLC) then quenched at -78 ° C.by dilution with ether (5 mL) and addition of saturated aqueous ammoniumchloride (0.3 mL). The mixture was allowed to warm to room temperatureand solid sodium sulfate was added to dry the solution. The mixture wasstirred 20 min, filtered, concentrated, and the resulting oil wasimmediately dissolved in acetonitrile (10 mL). To the solution of theresulting primary alcohol in CH₃ CN was added 2,6-lutidine (0.380 mL,3.3. mmol, 10 equiv) and N,N'-disuccinimidyl carbonate (420 mg, 1.65mmol, 5 equiv). The heterogenous mixture was stirred at room temperaturefor 19 h, at which time the solution was diluted with ether (30 mL) andwashed with saturated aqueous sodium bicarbonate (20 mL). The aqueousphase was back-extracted with ether (2×10 mL). The organic phases werecombined and dried (MgSO₄), concentrated, and subjected to flashchromatography (3:1 to 2:1 to 1:1 hexane/ethyl acetate). The desiredmixed carbonate 12 was isolated as a clear, colorless oil (217 mg, 0.184mmol, 56% overall for 4 steps).

Example 13

Preparation of 24, 24', 32, 32'-tetrakis(tert-butyldimethylsilyl)oxy!-FK1012-A

(p-xylylenediamine bridge) A dry, 1-mL conical glass vial was chargedwith the mixed carbonate (23.9 mg, 0.0203 mmol) and acetonitrile (500μL, 41 mM final concentration). Triethylamine (28 μL, 0.20 mmol, 10equiv) was added followed by p-xylylenediamine (46 μL, 0.0101 mmol,0.22M solution in DMF). The reaction stirred 18 h at room temperature,the solvent was removed with a stream of dry nitrogen, and the oil wasdirectly subjected to flash chromatography (3:1 to 2:1 to 1:1hexane/ethyl acetate) affording the desired protected dimer as a clear,colorless oil (11.9 mg, 5.3 μmol, 52%).

Example 14

Preparation of FK1012-A (p-xylylenediamine bridge) (13)

The protected dimer (11.0 mg, 4.9 μmol) was placed in a 1.5-mLpolypropylene tube fitted with a spin vane. Acetonitrile (0.50 mL, 10 mMfinal concentration) was added, and the solution stirred at 20° C. as HF(55 μL, 48% aqueous solution; Fisher, 3.0N final concentration) wasadded. The solution was stirred 16 h at room temperature. Thedeprotected FK506 derivative was then partitioned betweendichloromethane and saturated aqueous sodium bicarbonate in a 15-mL testtube. The tube was vortexed extensively to mix the phases and, afterseparation, the organic phase was removed with a pipet. The aqueousphase was back-extracted with dichloromethane (4×2 mL), and the combinedorganic phases were dried (MgSO₄), concentrated and subjected to flashchromatography (1:1:1 hexane/THF/ether to 1:1 THF/ether) providingFK1012-A as a clear, colorless oil (5.5 mg, 3.0 μmol, 63%).

Example 15

Preparation of 24, 24', 32,32'-tetrakis!(ter-butyldimethylsilyl)oxyl-FK1012-B (diaminodecanebridge)

A dry, 1-mL conical glass vial was charged with the mixed carbonate(53.3 mg, 0.0453 mmol) and acetonitrile (2.0 mL, 11 mM finalconcentration). Triethylamine (16 μL, 0.11 mmol, 5 equiv) was addedfollowed by diaminodecane (61 μL, 0.0226 mmol, 0.37M solution in DMF).The reaction stirred 12 h at room temperature, the solvent was removedwith a stream of dry nitrogen, and the oil was directly subjected toflash chromatography (3:1 to 2:1 to 1:1 hexane/ethyl acetate) affordingthe desired protected dimer as a clear, colorless oil (18.0 mg, 7.8μmol, 35%).

Example 16

Preparation of FK1012-B (diaminodecane-1,10 bridge) (14)

The protected dimer (18.0 mg, 7.8 μmol) was placed in a 1.5-mLpolypropylene tube fitted with a stirring flea. Acetonitrile (0.45 mL,16 mM final concentration) was added, and the solution stirred at roomtemperature as HF (55 μL, 48% aqueous solution; Fisher, 3.6N finalconcentration) was added. The solution was stirred 17 h at 23° C. Theproduct FK1012-B was then partitioned between dichloromethane andsaturated aqueous sodium bicarbonate in a 15-mL test tube. The tube wasvortexed extensively to mix the phases and, after separation, theorganic phase was removed with a pipet. The aqueous phase wasback-extracted with dichloromethane (4×2 mL), and the combined organicphases were dried (MgSO₄), concentrated and subjected to flashchromatography (100% ethyl acetate to 20:1 ethyl acetate/methanol)affording FK1012-B as a clear, colorless oil (5.3 mg, 2.9 μmol, 37%).

Example 17

Preparation of 24, 24', 32, 32'-tetrakis(tert-butyldimethylsilyl)oxy!-FK1012-C (his-p-aminomethylbenzoyldiaminodecane bridge)

A dry 25-mL tear-shaped flask was charged with the diamine linker (15.1mg, 0.0344 mmol) and 1.0 mL of DMF. In a separate flask, the mixedcarbonate and triethylamine (0.100 mL, 0.700 mmol, 20 equiv) weredissolved in 2.0 mL of dichloromethane then added slowly (4×0.50 mL) tothe stirring solution of his-p-aminomethylbenzoyl, diaminodecane -1,10.The flask containing the mixed carbonate 12 was washed withdichloromethane (2×0.50 mL) to ensure complete transfer of the mixedcarbonate 12. The reaction stirred 16 h at 23° C., the solvent wasremoved with a stream of dry nitrogen, and the oil was directlysubjected to flash chromatography (1:1 to 1:2 hexane/ethyl acetate) toafford the desired protected dimer as a clear, colorless oil (29.6 mg,11.5 μmol, 34%).

Example 18

Preparation of FK1012-C (15)

The protected dimer (29.6 mg, 11.5 μmol) (17) was placed in a 1.5-mLpolypropylene tube fitted with a stirring flea. Acetonitrile (0.45 mL,23 mM final concentration) was added, and the solution stirred at roomtemperature as HF (55 μL, 48% aqueous solution; Fisher, 3.6N finalconcentration) was added. The solution was stirred 17 h at roomtemperature. The desired symmetrical dimer was then partitioned betweendichloromethane and saturated aqueous sodium bicarbonate in a 15-mL testtube. The tube was vortexed extensively to mix the phases and, afterseparation, the organic phase was removed with a pipet. The aqueousphase was back-extracted with dichloromethane (4×2 mL), and the combinedorganic phases were dried (MgSO₄), concentrated and subjected to flashchromatography (100% ethyl acetate to 15:1 ethyl acetate/methanol)affording FK1012-C as a clear, colorless oil (11.5 mg, 5.5 μmol, 47%).

Preparation of CsA Derivatives

Example 19

MeBmt(OAc)-- --OH¹ CsA (2)

MeBmt(OAc)-- --OAc¹ -CsA (1) (161 mg, 124 mmol) (see Eberle andNuninger, J. Org. Chem. (1992) 57, 2689) was dissolved in Methanol (10mL). KOH (196 mg) was dissolved in water (8 mL). 297 mL of the KOHsolution (.130 mmol, 1.05 eq.) was added to the solution of (1) in MeOH.This new solution was stirred at room temperature under an inertatmosphere for 4 hours at which time the reaction was quenched withacetic acid (2 mL). The reaction mixture was purified by reversed phaseHPLC using a 5 cm×25 cm, 12μ, 100 A, C18 column at 70° C. eluting with70% acetonitrile/H₂ O containing 0.1% (v/v) Trifluoroacetic acid to give112 mg (72%) of the desired monoacetate (2).

MeBmt(OAc)-- --OCOIm¹ CsA (3)

MeBmt(OAc)-- --OH¹ -CsA (2) (57 mg, 45.5 μmol) and carbonyldiimidazole(15 mg, 2 eq., 91 μmol.) were transferred into a 50 mL round bottomflask and dissolved in dry THF (6 mL). Diisopropylethylamine (32 μL, 4eq., 182 μmol) was added and then the solvent was removed on a rotaryevaporator at room temperature. The residue was purified by flashchromatography on silica gel using ethyl acetate as eluent to give 45 mg(73%) of the desired carbamate (3).

Tris-(2-aminoethyl)amine CsA Trimer Triacetate (6)

MeBmt(OAc)-- --OCOIm¹ -CsA (3) (7.5 mg, 5.54 μmol, 3.1 eq.) wasdissolved in THF (100 μL). Diisopropylethylamine (62 μL, 5 eq., 8.93μmol of a solution containing 100 μL of amine in 4 mL THF) was addedfollowed by tris(2-aminoethyl)amine (26 μL, 1.79 μmol, 1 eq. of asolution containing 101 mg of tris-amine in 10 mL THF). This solutionwas allowed to stir under N₂ atmosphere for 5 days. The reaction mix wasevaporated and then purified by flash chromatography on silica gel using0-5% methanol in chloroform to give 4.1 mg of desired product (6).

Example 20

Diaminodecane CsA Dimer (8)

Solid Na metal (200 mg, excess) was reacted with dry methanol (10 mL) at0° C. Diaminodecane CsA Dimer Diacetate (5) (4.0 mg) was dissolved inMeOH (5 mL). 2.5 mL of the NaOMe solution was added to the solution of(5). After 2.5 hours of stirring at room temperature under an inertatmosphere, the solution was quenched with acetic acid (2 mL) and theproduct was purified by reversed phase HPLC using a 5 mm×25 mm, 12μ, 100A, C18 column at 70° C. eluting with 70-95% acetonitrile/H₂ O over 20minutes containing 0.1% (v/v) Trifluoroacetic acid to give 2.5 mg (60%)of the desired diol.

The diaminodecane CsA Dimer Diacetate (5) was prepared by replacing thetris(2-aminoethyl)amine with 0.45 eq. of 1,10-diaminodecane.

Example 21

p-Xylylenediamine CsA Dimer (4)

The p-xylene diamine CsA Dimer (4) was prepared by replacing thetris(2-aminoethyl)amino with 0.45 eq. of p-xylylene diamine.

Following procedures described in the literature other derivatives ofcyclophilin are prepared by linking at a site other than the 1(MeBmt 1)site.

Position 8 D-isomer analogues are produced by feeding the producingorganism with the D-amino analogue to obtain incorporation specificallyat that site. See Patchett, et al., J. Antibiotics (1992) 45, 943(β-MeSO)D-Ala⁸ -CsA); Traber, et al., ibid. (1989) 42, 591). Theposition 3 analogues are prepared by poly-lithiation/alkylation of CsA,specifically at the -carbon of Sac3. See Wenger, Transplant Proceeding(1986) 18, 213, supp. 5 (for cyclophilin binding and activity profiles,particularly D-MePhe³ -CsA); Seebach, U.S. Pat. No. 4,703,033, issuedOct. 27, 1987 (for preparation of derivatives).

Instead of cyclosporin A, following the above-described procedures,other naturally-occurring variants of CsA may be multimerized for use inthe subject invention.

Example 22

(A) Structure-Based Design and Synthesis of FK1012-"Bump" Compounds andFKBP12s with Compensatory Mutations

Substituents at C9 and CIO of FK506, which can be and have been accessedby synthesis, clash with a distinct set of FKBP12 sidechain residues.Thus, one class of mutant receptors for such ligands should containdistinct modifications, one creating a compensatory hole for the C10substituent and one for the C9 substituent. Carbon 10 was selectivelymodified to have either an N-acetyl or N-formyl group projecting fromthe carbon (vs. a hydroxyl group in FK506). The binding properties ofthese derivatives clearly reveal that these C10 bumps effectivelyabrogate binding to the native FKBP12. FIG. 23 depicts schemes for thesynthesis of FK506-type moieties containing additional C9 bumps. Byassembling such ligands with linker moieties of this invention one canconstruct HED and HOD (and antagonist) reagents for chimeric proteinscontaining corresponding binding domains bearing compensatory mutations.An illustrative HED reagent is depicted in FIG. 23 that containsmodifications at C9 and C10'.

This invention thus encompasses a class of FK-506-type compoundscomprising an FK-506-type moiety which contains, at one or both of C9and C10, a functional group comprising --OR, --R, --(CO)OR, --NH(CO)H or--NH(CO)R, where R is substituted or unsubstituted, alkyl or arylalkylwhich may be straightchain, branched or cyclic, including substituted orunsubstituted peroxides, and carbonates. "FK506-type moieties" includeFK506, FK520 and synthetic or naturally occurring variants, analogs andderivatives thereof (including rapamycin) which retain at least the(substituted or unsubstituted) C2 through C15 portion of the ringstructure of FK-506 and are capable of binding with a natural ormodified FKBP, preferably with a Kd value below about 10⁻⁶.

This invention further encompasses homo- and hetero-dimers and higherorder oligomers containing one or more of such FK-506-type compoundscovalently linked to a linker moiety of this invention. Monomers ofthese FK-506-type compounds are also of interest, whether or notcovalently attached to a linker moiety or otherwise modified withoutabolishing their binding affinity for the corresponding FKBP. Suchmonomeric compounds may be used as oligomerization antagonist reagents,i.e., as antagonists for oligomerizing reagents based on a likeFK-506-type compound. Preferably the compounds and oligomers comprisingthem in accordance with this invention bind to natural, or preferablymutant, FKBPs with an affinity at least 0.1% and preferably at leastabout 1% and even more preferably at least about 10% as great as theaffinity of FK506 for FKBP12. See e.g. Holt et al, infra.

Receptor domains for these and other ligands of this invention may beobtained by structure-based, site-directed or random mutagenesismethods. We contemplate a family of FKBP12 moieties which contain Val,Ala, Gly, Met or other small amino acids in place of one or more ofTyr26, Phe36, Asp37, Tyr82 and Phe99 as receptor domains for FK506-typeand FK-520-type ligands containing modifications at C9 and/or C10.

Site-directed mutagenesis may be conducted using the megaprimermutagenesis protocol (see e.g., Sakar and Sommer, BioTechniques 8 4(1990): 404-407). cDNA sequencing is performed with the Sequenase kit.Expression of mutant FKBP12s may be carried out in the plasmid pHN1⁺ inthe E. coli strain XA90 since many FKBP12 mutants have been expressed inthis system efficiently. Mutant proteins may be conveniently purified byfractionation over DE52 anion exchange resin followed by size exclusionon Sepharose as described elsewhere. See e.g. Aldape et al, J Biol Chem267 23 (1992): 16029-32 and Park et al, J Biol Chem 267 5 (1992):3316-3324. Binding constants may be readily determined by one of twomethods. If the mutant FKBPs maintain sufficient rotamase activity, thestandard rotamase assay may be utilized. See e.g., Galat et al,Biochemistry 31 (1992): 2427-2434. Otherwise, the mutant FKBP12s may besubjected to a binding assay using LH20 resin and radiolabeledT2-dihydroFK506 and T2-dihyroCsA that we have used previously with FKBPsand cyclophilins. Bierer et al, Proc. Natl. Acad. Sci. U.S.A. 87 4(1993): 555-69.

(B) Selection of Compensatory Mutations in FKBP12 for Bump-FK506s Usingthe Yeast Two-Hybrid System

One approach to obtaining variants of receptor proteins or domains,including of FKBP12, is the powerful yeast "two-hybrid" or "interactiontrap" system. The two-hybrid system has been used to detect proteinsthat interact with each other. A "bait" fusion protein consisting of atarget protein fused to a transcriptional activation domain isco-expressed with a cDNA library of potential "hooks" fused to aDNA-binding domain. A protein-protein (bait-hook) interaction isdetected by the appearance of a reporter gene product whose synthesisrequires the joining of the DNA-binding and activation domains. Theyeast two-hybrid system mentioned here was originally developed byElledge and co-workers. Durfee et al, Genes & Development 7 4 (1993):555-69 and Harper et al, Cell 75 4 (1993): 805-816.

Since the two-hybrid system per se cannot provide insights intoreceptor-ligand interactions involving small molecule, organic ligands,we have developed a new, FK1012-inducible transcriptional activationsystem (discussed below). Using that system one may extend the twohybrid system so that small molecules (e.g., FK506s or FK1012s orFK506-type molecules of this invention) can be investigated. One firstgenerates a cDNA library of mutant FKBPs (the hooks) with mutations thatare regionally localized to sites that surround C9 and C10 of FK506. Forthe bait, two different strategies may be pursued. The first uses theability of FK506 to bind to FKBP12 and create a composite surface thatbinds to calcineurin. The sequence-specific transcriptional activator isthus comprised of: DNA-binding domain-mutant FKBPI2 - - -bump-FK506 - - - calcineurin A-activation domain (where - - - refers toa noncovalent binding interaction). The second strategy uses the abilityof FK1012s to bind two FKBPs simultaneously. A HED version of an FK1012may be used to screen for the following ensemble: DNA-bindingdomain-mutant FKBP12---bump-FK506-normal FK506 - - - wildtypeFKBP12-activation domain.

1. Calcineurin-Gal4 Activation Domain Fusion as a Bait

A derivative of pSE1107 that contains the Gal4 activation domain andcalcineurin A subunit fusion construct has been constructed. Its abilityto act as a bait in the proposed manner has been verified by studiesusing the two-hybrid system to map out calcineurin's FKBP-FK506 bindingsite.

2. hFKBP12-Gal4 Activation Domain Fusion as a Bait

hFKBP12 cDNA may be excised as an EcoRI-HindIII fragment that covers theentire open reading frame, blunt-ended and ligated to the blunt-endedXho I site of pSE1107 to generate the full-length hFKBP-Gal4 activationdomain protein fusion.

3. Mutant hFKBP12 cDNA Libraries

hFKBP12 may be digested with EcoRI and HindIII, blunted and cloned intopAS1 (Durfee et al, supra) that has been cut with Ncol and blunted. Thisplasmid is further digested with NdeI to eliminate the NdeI fragmentbetween the Ndel site in the polylinker sequence of pAS1 and the 5' endof hFKBP12 and religated. This generated the hFKBP12-Gal4 DNA bindingdomain protein fusion. hFKBP was reamplified with primers #11206 SEQ IDNO:67! and #11210 SEQ ID NO:75!, Primer Table: ##STR9## Primer Table:SEQ ID NOS:67-76! Primers used in the construction of a regionallylocalized nFKBP12 cDNA library for use in screening for compensatorymutations.

Mutant hFKBP12 cDNA fragments were then prepared using the primerslisted below that contain randomized mutant sequences of hFKBP atdefined positions by the polymerase chain reaction, and were insertedinto the Gal4 DNA binding domain-hFKBP(NdeI/BamHI) construct.

4. Yeast strain

S. cerevisiae Y153 carries two selectable marker genes(his3/β-galctosidase) that are integrated into the genome and are drivenby Gal4 promoters. (Durfee, supra.)

Using Calcineurin-Gal4 Activation Domain as Bait

The FKBP12-FK506 complex binds with high affinity to calcineurin, a type2B protein phosphatase. Since we use C9- or C10-bumped ligands to serveas a bridge in the two-hybrid system, only those FKBPs from the cDNAlibrary that contain a compensatory mutation generate a transcriptionalactivator. For convenience, one may prepare at least three distinctlibraries (using primers 11207-11209, Primer Table) that will eachcontain 8,000 mutant FKBP12s. Randomized sites were chosen by inspectingthe FKBP12-FK506 structure, which suggested clusters of residues whosemutation might allow binding of the offending C9 or C10 substituents onbumped FK506s. The libraries are then individually screened using bothC9- and C10-bumped FK506s. The interaction between a bumped-FK506 and acompensatory hFKBP12 mutant can be detected by the ability of host yeastto grow on his drop-out medium and by the expression of β-galactosidasegene. Since this selection is dependent on the presence of thebumped-FK506, false positives can be eliminated by substractivescreening with replica plates that are supplemented with or without thebumped-FK506 ligands.

Using hFKBP12-Gal4 Activation Domain as Bait

Using the calcineurin A-Gal4 activation domain to screen hFKBP12 mutantcDNA libraries is a simple way to identify compensatory mutations onFKBP12. However, mutations that allow bumped-FK506s to bind hFKBP12 maydisrupt the interaction between the mutant FKBP12 - - - bumped-FK506complex and calcineurin. If the initial screening with calcineurin as abait fails, the wildtype hFKBP12-Gal4 activation domain will instead beused. An FK1012 HED reagent consisting of: native-FK506-bumped-FK506(FIG. 16) may be synthesized and used as a hook. The FK506 moiety of theFK102 can bind the FKBP12-Gal4 activation domain. An interaction betweenthe bumped-FK506 moiety of the FK1012 and a compensatory mutant ofFKBP12 will allow host yeast to grow on his drop-out medium and toexpress β-galactosidase. In this way, the selection is based solely onthe ability of hFKBP12 mutant to interact with the bumped-FK506. Thesame substractive screening strategy can be used to eliminate falsepositives.

In addition to the in vitro binding assays discussed earlier, an in vivoassay may be used to determine the binding affinity of the bumped-FK506sto the compensatory hFKBP12 mutants. In the yeast two-hybrid system,β-gal activity is determined by the degree of interaction between the"bait" and the "prey". Thus, the affinity between the bumped-FK506 andthe compensatory FKBP12 mutants can be estimated by the correspondingβ-galactosidase activities produced by host yeasts at different HED(native-FK506-bumped-FK506) concentrations.

Using the same strategy, additional randomized mutant FKBP12 cDNAlibraries may be created in other bump-contact residues withlow-affinity compensatory FKBP12 mutants as templates and may bescreened similarly.

Phage Display Screening for High-Affinity Compensatory FKBP Mutations

Some high-affinity hFKBP12 mutants for bump-FK506 may contain severalcombined point mutations at discrete regions of the protein. The size ofthe library that contains appropriate combined mutations can be toolarge for the yeast two-hybrid system's capacity (e.g., >10⁸ mutations).The use of bacteriophage as a vehicle for exposing whole functionalproteins should greatly enhance the capability for screening a largenumbers of mutations. See e.g. Bass et al, Proteins: Structure, Function& Genetics 8 4 (1990): 309-14; McCafferty et al, Nature 348 6301 (1990):552-4; and Hoogenboom, Nucl Acids Res 19 15 (1991): 4133-7. If thedesired high-affinity compensatory mutants is not be identified with theyeast two-hybrid system, a large number of combined mutations can becreated on hFKBP12 with a phage vector as a carrier. The mutant hFKBP12fusion phages can be screened with bumped-FK506-Sepharose as an affinitymatrix, which can be synthesized in analogy to our original FK506-basedaffinity matrices. Fretz et al, J Am Chem Soc 113 4 (1991): 1409-1411.Repeated rounds of binding and phage amplification should lead to theidentification of high-affinity compensatory mutants.

(C) Synthesis of "Bumped (CsA)2s": Modification of MeVal(11)CsA

As detailed above, we have demonstrated the feasibility of usingcyclophilin as a dimerization domain and (CsA)2 as a HOD reagent in thecontext of the cell death signaling pathway. However, to furtheroptimize the cellular activity of the (CsA)2 reagent one may rely uponsimilar strategies as described with FK1012s. Thus, modified (bumped)CsA-based oligomerizing reagents should be preferred in applicationswhere it is particularly desirable for the reagent to be able todifferentiate its target, the artificial protein constructs, fromendogenous cyclophilins.

One class of modified CsA derivatives of this invention are CsA analogsin which (a) NMeVal11 is replaced with NMePhe (which may be substitutedor unsubstituted) or NMeThr (which may be unsubstituted or substitutedon the threonine betahydroxyl group) or (b) the pro-S methyl group ofNMeVal11 is replaced with a bulky group of at least 2 carbon atoms,preferably three or more, which may be straight, branched and/or containa cyclic moiety, and may be alkyl (ethyl, or preferably propyl, butyl,including t-butyl, and so forth), aryl, or arylalkyl. These compoundsinclude those CsA analogs which contain NMeLeu, NMeIle, NMePhe orspecifically the unnatural NMe betaMePhe!, in place of MeVal11. The"(b)" CsA compounds are of formula 2 where R represents a functionalgroup as discussed above. ##STR10##

This invention further encompasses homo- and hetero-dimers and higherorder oligomers containg one or more such CsA analogs. Preferably thecompounds and oligomers comprising them in accordance with thisinvention bind to natural, or preferably mutant, cyclophilin proteinswith an affinity at least 0.1% and preferably at least about 1% and evenmore preferably at least about 10% as great as the affinity of CsA forcyclophilin.

A two step strategy may be used to prepare the modified MeVal¹¹ !CsAderivatives starting from CsA. In the first step the residue MeVal11 isremoved from the macrocycle. In the second step a selected amino acid isintroduced at the (former) MeVal11 site and the linear peptide iscyclized. The advantage of this strategy is the ready access to severalmodified MeVal¹¹ !CsA derivatives in comparison with a total synthesis.The synthetic scheme is as follows: ##STR11##

To differentiate the amide bonds, an N,O shift has been achieved betweenthe amino and the hydroxyl groups from MeBmt1 to give IsoCsA (Ruegger etal, Helv Chim Acta 59 4 (1976): 1075-92) (see scheme above). Thereaction was carried out in THF in the presence of methanesulfonic acid.(Oliyai et al, Pharm Res 9 5 (1992): 617-22). The free amine wasprotected with an acetyl group with pyridine and acetic anhydride in aone-pot procedure. The overall yield of the N-acetyl protected IsoCsA is90%. The ester MeBmt1-MeVal11 bond is then reduced selectively in thepresence of the N-methyl amide bonds, e.g. using DIBAL-H. The resultingdiol is then transformed to the corresponding di-ester with anotheracid-induced N,O shift. This will prepare both the N-acetyl group andMeVal11 residues for removal through hydrolysis of the newly formedesters with aqueous base.

After protection of the free amino group the new amino acid residue isintroduced e.g. with the PyBrop coupling agent. Deprotection andcyclization of the linear peptide with BOP in presence of DMAP (Albergand Schreiber, Science 262 5131 (1993): 248-250) completes the synthesisof 2. The binding of bumped-CsAs to cyclophilins can be evaluated by thesame methods described for FK506s and FK1012s. Once cyclophilins areidentified with compensatory mutations, bumped (CsA)2 HED and HODreagents may be synthesized according to the methods discussedpreviously. Of particular interest are bumped CsA compounds which canform dimers which themselves can bind to a cyclophilin protein with 1:2stoichiometry. Homo dimers and higher order homo-oligomers, heterodimersand hetero-higher order oligomers containing at least one such CsA ormodified CsA moiety may be designed and evaluated by the methodsdeveloped for FK1012A and (CsA)2, and optimize the linker element inanalogy to the FK1012 studies.

Mutant cyclophilins that bind our position 11 CsA variants (2) byaccomodating the extra bulk on the ligand may be now be prepared.Cyclophilins with these compensatory mutations may be identified throughthe structure-based site-directed and random mutagenesis/screeningprotocols described in the FK1012 studies.

It is evident from the above results, that the subject method andcompositions provide for great versatility in the production of cellsfor a wide variety of purposes. By employing the subject constructs, onecan use cells for therapeutic purposes, where the cells may remaininactive until needed, and then be activated by administration of a safedrug. Because cells can have a wide variety of lifetimes in a host,there is the opportunity to treat both chronic and acute indications soas to provide short- or long-term protection. In addition, one canprovide for cells which will be directed to a particular site, such asan anatomic site or a functional site, where therapeutic effect may beprovided.

Cells can be provided which will result in secretion of a wide varietyof proteins, which may serve to correct a deficit or inhibit anundesired result, such as activation of cytolytic cells, to inactivate adestructive agent, to kill a restricted cell population, or the like. Byhaving the cells present in the host over a defined period of time, thecells may be readily activated by taking the drug at a dose which canresult in a rapid response of the cells in the host. Cells can beprovided where the expressed chimeric receptor is intracellular,avoiding any immune response due to a foreign protein on the cellsurface. Furthermore, the intracellular chimeric receptor proteinprovides for efficient signal transduction upon ligand binding,apparently more efficiently than the receptor binding at anextracellular receptor domain.

By using relatively simple molecules which bind to chimeric membranebound receptors, resulting in the expression of products of interest orinhibiting the expression of products, one can provide for cellulartherapeutic treatment. The compounds which may be administered are safe,can be administered in a variety of ways, and can ensure a very specificresponse, so as not to upset homeostasis.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 81    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    MetGlySerSerLysSerLysProLysAspProSerGlnArg    1510    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    GTTAAGTTAAC11    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    TGACTCAGCGC11    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 33 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 6..11    (D) OTHER INFORMATION: /note= "Sac II restriction site."    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- signal    (B) LOCATION: 12..16    (D) OTHER INFORMATION: /note= "Kozak sequence."    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 17..31    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 17..33    (D) OTHER INFORMATION: /note= "Region of homology with    target sequence."    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    CGACACCGCGGCCACCATGGCCACAATTGGAGC33    MetAlaThrIleGly    15    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 5 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    MetAlaThrIleGly    15    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 6..11    (D) OTHER INFORMATION: /note= "Xho I restriction site."    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 12..27    (D) OTHER INFORMATION: /note= "Region of homology with    target sequence."    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    CGACACTCGAGAGCCCATGACTTCTGG27    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..4    (D) OTHER INFORMATION: /note= "Translation product of    complement of SEQ ID NO:6, bases 9 to 20."    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    SerTrpAlaLeu    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 41 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 6..11    (D) OTHER INFORMATION: /note= "Xho I restriction site."    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 12..41    (D) OTHER INFORMATION: /note= "Region of homology with    target sequence."    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 9..41    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 28    (D) OTHER INFORMATION: /note= "A to G."    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    CGACACTCGAGCTCTGCTACTTGCTAGGTGGAATCCTCTTC41    GluLeuCysTyrLeuLeuGlyGlyIleLeuPhe    1510    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    GluLeuCysTyrLeuLeuGlyGlyIleLeuPhe    1510    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 3..8    (D) OTHER INFORMATION: /note= "Eco RI restriction site."    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 9..24    (D) OTHER INFORMATION: /note= "Region of homology with    target sequence."    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 24    (D) OTHER INFORMATION: /note= "G to C."    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- signal    (B) LOCATION: complement (9..11)    (D) OTHER INFORMATION: /note= "Translational stop encoded    in complementary strand."    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    GCGAATTCTTAGCGAGGGGCCAGC24    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..4    (D) OTHER INFORMATION: /note= "Translational product of    complement to SEQ ID NO:10, bases 12 to 23."    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    LeuAlaProArg    1    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 33 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 3..8    (D) OTHER INFORMATION: /note= "Eco RI restriction."    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 12..17    (D) OTHER INFORMATION: /note= "Sal I restriction site."    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- signal    (B) LOCATION: complement (9..11)    (D) OTHER INFORMATION: /note= "Translational stop signal    encoded on complementary strand."    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 18..33    (D) OTHER INFORMATION: /note= "Region of homology with    target sequence."    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    GCGAATTCTTAGTCGACGCGAGGGGCCAGGGTC33    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..4    (D) OTHER INFORMATION: /note= "Translational product of    complement to SEQ ID NO:12, bases 18 to 29."    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    LeuAlaProArg    1    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 25 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 4..9    (D) OTHER INFORMATION: /note= "Xho I restriction site."    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 13    (D) OTHER INFORMATION: /note= "T to G."    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 4..25    (D) OTHER INFORMATION: /note= "Region of homology with    target sequence."    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 10..24    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    GGGCTCGAGCTCGGCTACTTGCTAG25    LeuGlyTyrLeuLeu    15    (2) INFORMATION FOR SEQ ID NO:15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 5 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    LeuGlyTyrLeuLeu    15    (2) INFORMATION FOR SEQ ID NO:16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 6..11    (D) OTHER INFORMATION: /note= "Xho I restriction site."    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 12..26    (D) OTHER INFORMATION: /note= "Region of homology with    target sequence."    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    CGACACTCGAGGTGACGGACAAGGTC26    (2) INFORMATION FOR SEQ ID NO:17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 6..11    (D) OTHER INFORMATION: /note= "Sal I restriction site."    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 12..26    (D) OTHER INFORMATION: /note= "Region of homology with    target sequence."    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    CGACAGTCGACCCAATCAGGGACCTC26    (2) INFORMATION FOR SEQ ID NO:18:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 33 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..5    (D) OTHER INFORMATION: /note= "Xho I restriction site."    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 10..15    (D) OTHER INFORMATION: /note= "Bsi WI restriction site."    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 6..32    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    TCGAGTATCCGTACGACGTACCAGACTACGCAG33    TyrProTyrAspValProAspTyrAla    15    (2) INFORMATION FOR SEQ ID NO:19:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 9 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    TyrProTyrAspValProAspTyrAla    15    (2) INFORMATION FOR SEQ ID NO:20:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 33 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..5    (D) OTHER INFORMATION: /note= "Sal I restriction site."    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:    TCGACTGCGTAGTCTGGTACGTCGTACGGATAC33    (2) INFORMATION FOR SEQ ID NO:21:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 33 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..5    (D) OTHER INFORMATION: /note= "Sal I restriction site."    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:    TCGACTATCCGTACGACGTACCAGACTACGCAC33    (2) INFORMATION FOR SEQ ID NO:22:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 33 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..5    (D) OTHER INFORMATION: /note= "Xho I restriction site."    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:    TCGAGTGCGTAGTCTGGTACGTCGTACGGATAG33    (2) INFORMATION FOR SEQ ID NO:23:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 80 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 6..11    (D) OTHER INFORMATION: /note= "Sac II restriction site."    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- signal    (B) LOCATION: 12..16    (D) OTHER INFORMATION: /note= "Kozak sequence."    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- signal    (B) LOCATION: 17..58    (D) OTHER INFORMATION: /note= "Myristoylation signal."    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 59..64    (D) OTHER INFORMATION: /note= "Xho I restriction site."    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 65..80    (D) OTHER INFORMATION: /note= "Zeta homology."    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 17..79    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:    CGACACCGCGGCCACCATGGGGAGTAGCAAGAGCAAGCCTAAGGACCCC49    MetGlySerSerLysSerLysProLysAspPro    1510    AGCCAGCGCCTCGAGAGGAGTGCAGAGACTG80    SerGlnArgLeuGluArgSerAlaGluThr    1520    (2) INFORMATION FOR SEQ ID NO:24:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:    MetGlySerSerLysSerLysProLysAspProSerGlnArgLeuGlu    151015    ArgSerAlaGluThr    20    (2) INFORMATION FOR SEQ ID NO:25:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 12..26    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 6..11    (D) OTHER INFORMATION: /note= "Xho I restriction site."    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 12..27    (D) OTHER INFORMATION: /note= "Region of homology with    target sequence."    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:    CGACACTCGAGGAGCTCTGTGACGATG27    GluLeuCysAspAsp    15    (2) INFORMATION FOR SEQ ID NO:26:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 5 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:    GluLeuCysAspAsp    15    (2) INFORMATION FOR SEQ ID NO:27:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 41 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 6..11    (D) OTHER INFORMATION: /note= "Xho I restriction site."    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 12..41    (D) OTHER INFORMATION: /note= "Region of homology with    target sequence."    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 27..29    (D) OTHER INFORMATION: /note= "GAT to AAG."    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 9..41    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:    CGACACTCGAGCTCTGCTACTTGCTAAAGGGAATCCTCTTC41    GluLeuCysTyrLeuLeuLysGlyIleLeuPhe    1510    (2) INFORMATION FOR SEQ ID NO:28:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:    GluLeuCysTyrLeuLeuLysGlyIleLeuPhe    1510    (2) INFORMATION FOR SEQ ID NO:29:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 44 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 6..11    (D) OTHER INFORMATION: /note= "Xho I restriction site."    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 9..44    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 27..44    (D) OTHER INFORMATION: /note= "Region of homology with target    sequence."    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:    CGACACTCGAGCTGCTGGATCCGAAGCTCTGCTACTTGCTAAAG44    GluLeuLeuAspProLysLeuCysTyrLeuLeuLys    1510    (2) INFORMATION FOR SEQ ID NO:30:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:    GluLeuLeuAspProLysLeuCysTyrLeuLeuLys    1510    (2) INFORMATION FOR SEQ ID NO:31:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 31 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 6..11    (D) OTHER INFORMATION: /note= "Xho I restriction site."    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 12..31    (D) OTHER INFORMATION: /note= "Region of homology with    target sequence."    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 9..31    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:    CGACACTCGAGACAACAGAGTACCAGGTAGC31    GluThrThrGluTyrGlnValAla    15    (2) INFORMATION FOR SEQ ID NO:32:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 8 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:    GluThrThrGluTyrGlnValAla    15    (2) INFORMATION FOR SEQ ID NO:33:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 28 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 6..11    (D) OTHER INFORMATION: /note= "Xho I restriction site."    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 12..28    (D) OTHER INFORMATION: /note= "Region of homology with    target sequence."    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 9..28    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:    CGACACTCGAGGGCGTGCAGGTGGAGAC28    GluGlyValGlnValGluThr    15    (2) INFORMATION FOR SEQ ID NO:34:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 7 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:    GluGlyValGlnValGluThr    15    (2) INFORMATION FOR SEQ ID NO:35:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 6..11    (D) OTHER INFORMATION: /note= "Sal I restriction site."    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 12..27    (D) OTHER INFORMATION: /note= "Region of homology with    target sequence."    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: complement (9..26)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:    CGACAGTCGACTTCCAGTTTTAGAAGC27    (2) INFORMATION FOR SEQ ID NO:36:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 6 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:    LeuLeuLysLeuGluVal    15    (2) INFORMATION FOR SEQ ID NO:37:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 7..12    (D) OTHER INFORMATION: /note= "Xho I restriction site."    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 10..27    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 13..27    (D) OTHER INFORMATION: /note= "Region of homology with    target sequence."    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:    TCGACACTCGAGACGGGGGCCGAGGGC27    GluThrGlyAlaGluGly    15    (2) INFORMATION FOR SEQ ID NO:38:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 6 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:    GluThrGlyAlaGluGly    15    (2) INFORMATION FOR SEQ ID NO:39:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 28 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 7..12    (D) OTHER INFORMATION: /note= "Sal I restriction site."    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: complement (10..18)    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 13..28    (D) OTHER INFORMATION: /note= "Region of homology with    target sequence."    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:    CCGACAGTCGACCTCTATTTTGAGCAGC28    (2) INFORMATION FOR SEQ ID NO:40:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 3 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:    IleGluVal    1    (2) INFORMATION FOR SEQ ID NO:41:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 38 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:    CGACACCGCGGCCACCATGAAGCTACTGTCTTCTATCG38    (2) INFORMATION FOR SEQ ID NO:42:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 28 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:    CGACAGTCGACCGATACAGTCAACTGTC28    (2) INFORMATION FOR SEQ ID NO:43:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 38 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 6..11    (D) OTHER INFORMATION: /note= "Sac II restriction site."    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- signal    (B) LOCATION: 12..16    (D) OTHER INFORMATION: /note= "Kozak sequence."    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 17..37    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 17..38    (D) OTHER INFORMATION: /note= "Ga14 (1-147) coding region."    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:    CGACACCGCGGCCACCATGAAGCTACTGTCTTCTATCG38    MetLysLeuLeuSerSerIle    15    (2) INFORMATION FOR SEQ ID NO:44:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 7 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:    MetLysLeuLeuSerSerIle    15    (2) INFORMATION FOR SEQ ID NO:45:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 28 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..17    (D) OTHER INFORMATION: /note= "Region encoding for C-terminal    end of Ga14 (1- 147)."    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 3..17    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 18..23    (D) OTHER INFORMATION: /note= "Sal I restriction site."    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:    GACAGTTGACTGTATCGGTCGACTGTCG28    ArgGlnLeuThrValSer    15    (2) INFORMATION FOR SEQ ID NO:46:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 6 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:    ArgGlnLeuThrValSer    15    (2) INFORMATION FOR SEQ ID NO:47:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 34 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:    CGACACCGCGGCCACCATGGTTTCTAAGCTGAGC34    (2) INFORMATION FOR SEQ ID NO:48:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 28 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:    CGACAGTCGACCAACTTGTGCCGGAAGG28    (2) INFORMATION FOR SEQ ID NO:49:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 34 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 6..11    (D) OTHER INFORMATION: /note= "Sac II restriction site."    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- signal    (B) LOCATION: 12..16    (D) OTHER INFORMATION: /note= "Kozak sequence."    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 17..34    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 17..34    (D) OTHER INFORMATION: /note= "Region encoding N-terminal    end of HNF1 (1281)."    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:    CGACACCGCGGCCACCATGGTTTCTAAGCTGAGC34    MetValSerLysLeuSer    15    (2) INFORMATION FOR SEQ ID NO:50:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 6 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:    MetValSerLysLeuSer    15    (2) INFORMATION FOR SEQ ID NO:51:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 28 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..20    (D) OTHER INFORMATION: /note= "Region encoding for C-terminal    end of HNF1 (1- 282)."    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 3..17    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:    CCTTCCGGCACAAGTTGGTCGACTGTCG28    AlaPheArgHisLysLeu    15    (2) INFORMATION FOR SEQ ID NO:52:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 6 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:    AlaPheArgHisLysLeu    15    (2) INFORMATION FOR SEQ ID NO:53:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- signal    (B) LOCATION: 3..7    (D) OTHER INFORMATION: /note= "Kozak sequence."    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..11    (D) OTHER INFORMATION: /note= "Complementary to bases 5 to    15 of SEQ ID NO:54."    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:    GGCCACCATGC11    (2) INFORMATION FOR SEQ ID NO:54:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 3 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..3    (D) OTHER INFORMATION: /note= "Translation product of SEQ    ID NO:53 and SEQ ID NO:55. Translational    start site at base 8 of SEQ ID NO:53."    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:    MetLeuGlu    1    (2) INFORMATION FOR SEQ ID NO:55:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 14..17    (D) OTHER INFORMATION: /note= "Sac II restriction site    overhang."    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..5    (D) OTHER INFORMATION: /note= "Xho I restriction site    overhang."    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 5..15    (D) OTHER INFORMATION: /note= "Complementary to bases 1 to 11    of SEQ ID NO:53."    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:    TCGAGCATGGTGGCCGC17    (2) INFORMATION FOR SEQ ID NO:56:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:    TCGACCCTAAGAMGAAGAGAAAGGTAC27    (2) INFORMATION FOR SEQ ID NO:57:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:    TCGAGTACCTTTCTCTTCKTCTTAGGG27    (2) INFORMATION FOR SEQ ID NO:58:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..5    (D) OTHER INFORMATION: /note= "Sal I restriction site    overhang."    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 5..27    (D) OTHER INFORMATION: /note= "Complementary to SEQ ID NO:60,    bases 5 to 27."    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:    TCGACCCTAAGAAGAAGAGAAAGGTAC27    (2) INFORMATION FOR SEQ ID NO:59:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ix) FEATURE:    (A) NAME/KEY: Peptide    (B) LOCATION: 1..11    (D) OTHER INFORMATION: /note= "Translation product of SEQ ID    NOS:58 and 60."    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:    LeuAspProLysLysLysArgLysValLeuGlu    1510    (2) INFORMATION FOR SEQ ID NO:60:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..5    (D) OTHER INFORMATION: /note= "Xho I restriction site    overhang."    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 5..27    (D) OTHER INFORMATION: /note= "Complementary to SEQ ID NO:58,    bases 5 to 27."    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:    TCGAGTACCTTTCTCTTCTTCTTAGGG27    (2) INFORMATION FOR SEQ ID NO:61:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 29 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:    CGACAGTCGACGCCCCCCCGACCGATGTC29    (2) INFORMATION FOR SEQ ID NO:62:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:    CGACACTCGAGCCCACCGTACTCGTC26    (2) INFORMATION FOR SEQ ID NO:63:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 29 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 6..11    (D) OTHER INFORMATION: /note= "Sal I restriction site."    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 12..29    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 12..29    (D) OTHER INFORMATION: /note= "Region encoding Nterminal    end of VP16 (413490)."    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:    CGACAGTCGACGCCCCCCCGACCGATGTC29    AlaProProThrAspVal    15    (2) INFORMATION FOR SEQ ID NO:64:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 6 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:    AlaProProThrAspVal    15    (2) INFORMATION FOR SEQ ID NO:65:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..15    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..15    (D) OTHER INFORMATION: /note= "Region encoding C-terminal    end of VP16 (413-490)."    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:    GACGAGTACGGTGGGCTCGAGTGTCG26    AspGluTyrGlyGly    15    (2) INFORMATION FOR SEQ ID NO:66:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 5 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:    AspGluTyrGlyGly    15    (2) INFORMATION FOR SEQ ID NO:67:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 23 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:    GGAATTCCATATGGGCGTGCAGG23    (2) INFORMATION FOR SEQ ID NO:68:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 5 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:    HisMetGlyValGln    15    (2) INFORMATION FOR SEQ ID NO:69:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 39 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:    CTGTCCCGGGANNNNNNNNNTTTCTTTCCATCTTCAAGC39    (2) INFORMATION FOR SEQ ID NO:70:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:    ArgSerXaaXaaXaaLysLysGlyAspGluLeu    1510    (2) INFORMATION FOR SEQ ID NO:71:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 64 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:    CTGTCCCGGGAGGAATCAAATTTCTTTCCATCTTCAAGCATNNNNNNNNNGTGCACCACG60    CAGG64    (2) INFORMATION FOR SEQ ID NO:72:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:    ArgSerSerAspPheLysLysGlyAspGluLeuMetXaaXaaXaaHis    151015    ValValCys    (2) INFORMATION FOR SEQ ID NO:73:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 57 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:    CGCGGATCCTCATTCCAGTTTTAGAAGCTCCACATCNNNNNNNNNAGTGGCATGTGG57    (2) INFORMATION FOR SEQ ID NO:74:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:    GluLeuLysLeuLeuGluValAspXaaXaaXaaThrAlaHisPro    151015    (2) INFORMATION FOR SEQ ID NO:75:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 28 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:    CGCGGATCCTCATTCCAGTTTTAGAAGC28    (2) INFORMATION FOR SEQ ID NO:76:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 5 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:    GluLeuLysLeuLeu    15    (2) INFORMATION FOR SEQ ID NO:77:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 28 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:    CGACAGTCGACCGATACAGTCAACTGTC28    (2) INFORMATION FOR SEQ ID NO:78:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 28 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:    CGACAGTCGACCAACTTGTGCCGGAAGG28    (2) INFORMATION FOR SEQ ID NO:79:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:    TCGAGCATGGTGGCCGC17    (2) INFORMATION FOR SEQ ID NO:80:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:    TCGAGTACCTTTCTCTTCTTCTTAGGG27    (2) INFORMATION FOR SEQ ID NO:81:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:    CGACACTCGAGCCCACCGTACTCGTC26    __________________________________________________________________________

We claim:
 1. A genetic construct encoding a chimeric protein comprising(a) at least one ligand-binding domain which binds to a selected ligandso as to oligomerize two or more chimeric protein molecules to form aligand cross-linked protein complex, the selected ligand having one ormore of the following characteristics:(i) the ligand is not a protein;(ii) the ligand has a molecular weight less than 5 kD; and (iii) theligand is membrane permeable; and(b) an action domain, which isheterologous with respect to the ligand-binding domain, and whichinduces a biological process as a result of formation of the ligandcross-linked protein complex.
 2. The genetic construct of claim 1 whichencodes a chimeric protein comprising two or more ligand-bindingdomains.
 3. The genetic construct of claim 1 which encodes a chimericprotein comprising at least one ligand-binding domain which binds to aligand having a molecular weight less than 3 kD.
 4. The geneticconstruct of claim 1 which encodes a chimeric protein comprising atleast one ligand-binding domain having between 50 and 350 amino acidresidues.
 5. The genetic construct of claim 1 which encodes a chimericprotein comprising at least one naturally-occurring ligand-bindingdomain.
 6. The genetic construct of claim 1 which encodes a chimericprotein comprising at least one ligand-binding domain comprising anon-naturally-occurring peptide sequence.
 7. The genetic construct ofclaim 1 which encodes a chimeric protein which binds to the selectedligand with a Kd value less than or equal to about 10⁻⁶ M.
 8. Thegenetic construct of claim 1 which encodes a chimeric protein comprisingat least one ligand-binding domain comprising an antibiotic bindingdomain or antibody domain.
 9. The genetic construct of claim 1 whichencodes a chimeric protein comprising at least one ligand-binding domainwhich binds to FK506, FK520, or derivatives thereof.
 10. The geneticconstruct of claim 1 which encodes a chimeric protein comprising acytoplasmic domain of a cell surface receptor or mutein thereofsufficient to induce a biological process in the cell upon formation ofthe ligand cross-linked protein complex.
 11. The genetic construct ofclaim 1 wherein the action domain of the chimeric protein induces, uponoligomerization, gene transcription from a gene comprising a DNA elementselected from the group consisting of a cAMP responsive element, an SRE,a VL30, an RSRF, an ISRE, a GAS, an ARRE-1 and an ARRE-2.
 12. Thegenetic construct of claim 1, wherein the chimeric protein furthercomprises an intracellular localizing domain which directs the chimericprotein to a given cellular location.
 13. A composition comprising atleast one genetic construct of claim 1 encoding a chimeric protein and asecond genetic construct comprising a target gene under thetranscriptional control of a transcriptional control element responsiveto the oligomerization of the chimeric protein.
 14. The geneticconstruct of claim 1, wherein said ligand is a synthetic organicmolecule having a molecular weight of less than 5 kDa.
 15. The geneticconstruct of claim 1, wherein said ligand is membrane permeable.
 16. Thegenetic construct of claim 1, wherein said ligand comprises amacrocyclic compound.
 17. The genetic construct of claim 1 wherein theaction domain includes a cytoplasmic domain, or a subunit or portionthereof which induces a detectable biological response, of a receptorselected from the group consisting of CD3z, CD3η, CD3g, CD3d, CD3e, aninterferon receptor, an interleukin receptor, a GM-CSF receptor, a LIFreceptor, a CNTF receptor, an oncostatin M receptor, a TGF-β receptor,an EGF receptor, ATR2/neu, a HER2/neu, a HER3/c-erbB-3, Xmrk, an insulinreceptor, an IGF-1 receptor, IRR, PDGF receptor, a CSF-1 receptor,c-kit, STK-1/flk-2, an FGF receptor, flg, bek, an NGF receptor,Ig-alpha/MB-1, Ig-beta/B29, Ror1 and Ror2.
 18. The genetic construct ofclaim 2 which encodes a chimeric protein comprising three or moreligand-binding domains.
 19. The genetic construct of claim 4 whichencodes a chimeric protein comprising at least one ligand-binding domainhaving less than 200 amino acid residues.
 20. The genetic construct ofclaim 7 which encodes a chimeric protein which binds to the selectedligand with a Kd value less than or equal to about 10⁻⁸ M.
 21. Thegenetic construct of claim 9 which comprises at least one ligand-bindingdomain comprising an FK506 Binding Protein ("FKBP")12 or a variantthereof in which one or more of Tyr26, Phe36, Asp37, Try82 and Phe99 arereplaced with a different amino acid.
 22. The genetic construct of claim9 which comprises at least one ligand-binding domain comprising an FK506Binding Protein ("FKBP")12 or a variant thereof in which one or more ofTyr26, Phe36, Asp37, Try82 and Phe99 are replaced with different aminoacids independently selected from Val, Ala, Gly and Met.
 23. The geneticconstruct of claim 10 which encodes a chimeric protein comprising anaction domain derived from the cytoplasmic domain of a cell surfacereceptor selected from the group consisting of a tyrosine kinasereceptor, a cytokine receptor and a growth factor receptor.
 24. A cellcomprising the genetic construct of claim
 10. 25. The genetic constructof claim 12 in which the intracellular localizing domain comprises asecretory leader sequence, a membrane retention domain, a nuclearlocalization domain or a vesicle targeting domain.
 26. A cell comprisingthe genetic construct of claim
 12. 27. The composition of claim 13,wherein the target gene encodes a surface membrane protein, a secretedprotein, a cytoplasmic protein, an antisense message or a ribozyme. 28.The composition of claim 13, wherein the target gene encodes a hormone,growth factor, interleukin, enzyme or surface membrane protein.
 29. Thegenetic construct of claim 16, wherein the macrocyclic compound is amacrolide.
 30. The genetic construct of claim 22 which comprises atleast one FKBP12 variant in which Phe36 or Asp37 or both are replacedwith a different amino acid independently selected from Val or Ala. 31.The genetic construct of claim 22 which comprises at least one FKBP12variant in which Phe36 is replaced with valine.
 32. The geneticconstruct of claim 25 in which the membrane retention domain comprises amembrane spanning or lipid membrane binding domain.
 33. The geneticconstruct of any of claims 1, 21, 8, 4, 5, 6, 7, 8 10, 11, 12, 16, 29 or17, which encodes a chimeric protein comprising as an intracellulardomain at least one ligand-binding domain.
 34. The genetic construct ofany of claims 1, 2, 18, 4, 5, 6, 7, 8, 11, 12, 16 and 29 wherein theaction domain of the chimeric protein comprises:(a) a DNA bindingdomain; (b) a transcriptional activation domain; (c) a transcriptionalrepressor domain; or (d) a signaling domain which induces a detectablebiological process following oligomerization of the chimeric proteinmolecule to form the ligand cross-linked protein complex.
 35. Thegenetic construct of any of claims 1, 2, 18, 4, 5, 6, 7, 8, 10, 11, 12,16, 29 or 17, wherein the ligand-binding domain is a ligand-bindingdomain of an intracellular protein.
 36. A cell comprising the geneticconstruct of claim
 32. 37. The genetic construct of claim 34 whichencodes a chimeric protein which upon oligomerization with anotherchimeric protein triggers a detectable activity comprising proteinkinase activity, phosphatase activity, reductase activity,cyclooxygenase activity or protease activity.
 38. The genetic constructof claim 34 wherein the action domain of the chimeric protein, providedin the ligand cross-linked protein complex, induces a biological processselected from the group consisting of channel opening, ion release,acylation, methylation, hydrolysis, phosphorylation, dephosphorylation,change in redox states, and rearrangement reactions.
 39. The cellcomprising and the genetic construct of claim
 34. 40. A compositioncomprising (a) two genetic constructs of claim 34 which encode a pair ofchimeric proteins which bind to a common selected ligand and form anoligomeric ligand-crosslinked complex, and (b) an additional geneticconstruct comprising a target gene under the transcriptional control ofa transcriptional control element responsive to the oligomerization ofthe chimeric proteins.
 41. The genetic construct of claim 34, wherein atleast one ligand-binding domain is an intracellular domain of thechimeric protein.
 42. The genetic construct of claim 34, wherein atleast one ligand-binding domain is a domain from an intracellularprotein.
 43. The genetic construct of claim 34, wherein the chimericprotein, upon oligomerization to form the ligand cross-linked proteincomplex, induces cell death.
 44. The genetic construct of claim 38,wherein the chimeric protein, upon oligomerization to form the ligandcross-linked protein complex, induces gene transcription.
 45. A cellcomprising and the genetic construct of claim
 43. 46. A cell comprisingand the genetic construct of claim
 44. 47. A vector comprising a geneticconstruct encoding a chimeric protein comprising(a) at least oneligand-binding domain which binds to a selected ligand so as tooligomerize two or more chimeric protein molecules to form a ligandcross-linked protein complex, the selected ligand having one or more ofthe following characteristics:(i) the ligand is not a protein, (ii) theligand has a molecular weight less than 5 kD, and (iii) the ligand ismembrane permeable; and (b) an action domain which is heterologous withrespect to the ligand-binding domain, and which induces a biologicalprocess as a result of the formation of the ligand cross-linked proteincomplex,which vector further includes one or more of a bacterial oryeast origin of replication, a selectable marker, an amplifiable marker,a promoter, or an enhancer element for expression of the chimericprotein in procaryotic or eucaryotic cells.
 48. The vector of claim 47in which the genetic construct is expressed in a cell-specific manner.49. The vector of claim 47, comprising a promoter for expression of thechimeric protein in a eucarvotic cell.
 50. The vector of claim 47,wherein the vector is a viral vector.
 51. The vector of claim 47,wherein the genetic construct encodes a chimeric protein comprising twoor more ligand-binding domains.
 52. The vector of claim 47, wherein thegenetic construct encodes a chimeric protein comprising at least oneligand-binding domain which binds to a ligand having a molecular weightless than 3 kD.
 53. The vector of claim 47, wherein the geneticconstruct encodes a chimeric protein comprising at least oneligand-binding domain having between 50 and 350 amino acid residues. 54.The vector of claim 47 wherein the genetic construct encodes a chimericprotein comprising at least one naturally occurring ligand bindingdomain.
 55. The vector of claim 47 wherein the genetic construct encodesa chimeric protein comprising at least one ligand-binding domaincomprising a non-naturally-occurring peptide sequence.
 56. The vector ofclaim 47 wherein the genetic construct encodes a chimeric protein whichbinds to the selected ligand with a kD value less than or equal to about10⁻⁶ M.
 57. The vector of claim 47 wherein the genetic construct encodesa chimeric protein comprising at least one ligand binding domaincomprising an immunophilin domain, cyclophilin domain, steroid bindingdomain, antibiotic binding domain, or antibody domain.
 58. The vector ofclaim 47, wherein said ligand comprises a macrocyclic compound.
 59. Thevector of claim 47 wherein the genetic construct encodes a chimericprotein comprising at least one ligand-binding domain which binds toFK506, FK520, rapamycin, or derivatives thereof.
 60. The vector of claim47, wherein said ligand is a synthetic organic molecule having amolecular weight of less than 5 kDa.
 61. The vector of claim 47, whereinsaid ligand is membrane permeable.
 62. The vector of claim 47, whereinthe action domain induces a biological process selected from the groupconsisting of channel opening, ion release, acylation, methylation,hydrolysis, phosphorylation, dephosphorylation, change in redox states,and rearrangement reactions.
 63. The vector of claim 47 wherein theaction domain includes a cytoplasmic domain, or a portion thereof whichinduces a biological process, of a transmembrane receptor protein. 64.The vector of claim 47, wherein the action domain has an enzymaticactivity selected from the group consisting of a phosphatase activity, areductase activity, a cyclooxygenase activity and a protease activity.65. The vector of claim 47, wherein the genetic construct encodes achimeric protein comprising an intracellular localizing domain whichdirects the chimeric protein to a given cellular location.
 66. Thevector of claim 47, wherein the heterologous protein domain includes acytoplasmic domain, or a subunit or portion thereof which induces adetectable biological response, of a receptor selected from the groupconsisting of CD3ζ, CD3η, CD3γ, CD3δ, CD3ε, an interferon receptor, aninterleukin receptor, a GM-CSF receptor, a LIF receptor, a CNTFreceptor, an oncostatin M receptor, a TGF-β receptor, an EGF receptor,ATR2/neu, a HER2/neu, a HER3/c-erbB-3, Xmrk, an insulin receptor, anIGF-1 receptor, IRR, PDGF receptor, a CSF-1 receptor, c-kit,STK-1/flk-2, an FGF receptor, flg, bek, an NGF receptor, Ig-alpha/MB-1,Ig-beta/B29, Ror1 and Ror2.
 67. The vector of claim 47, wherein theaction domain is derived from a cytoplasmic domain of a cell surfacereceptor selected from the group consisting of a tyrosine kinasereceptor, a cytokine receptor and a growth factor receptor.
 68. Thevector of claim 47, wherein the action domain of the chimeric proteininduces, upon oligomerization, gene transcription from a gene comprisinga DNA element selected from the group consisting of a cAMP responsiveelement, an SRE, a VL30, an RSRF, an ISRE, a GAS, an ARRE-1 and anARRE-2.
 69. The vector of claim 50, wherein the viral vector is anadenoviral vector.
 70. The vector of claim 51, wherein the geneticconstruct encodes a chimeric protein comprising two or more differentligand-binding domains.
 71. The vector of claim 53, wherein the geneticconstruct encodes a chimeric protein comprising at least one ligandbinding domain having less than 200 amino acid residues.
 72. The vectorof claim 56 wherein the genetic construct encodes a chimeric proteinwhich binds to the selected ligand with a kD value less than or equal toabout 10⁻⁸ M.
 73. The vector of claim 58, wherein the macrocycliccompound is a macrolide.
 74. The vector of claim 59 wherein the chimericprotein comprises at least one ligand-binding domain comprising an FK506binding protein ("FKBP") 12 or a variant thereof in which one or more ofTyrZ6, Phe36, Asp37, Try82, and Phe99 are replaced with a differentamino acid.
 75. The vector of claim 59 wherein the chimeric proteincomprises at least one ligand-binding domain comprising an FKBP12 or avariant thereof in which one or more of Tyr26, Phe36, Asp37, Try82, andPhe99 are replaced with different amino acids independently selectedfrom Val, Ala, Gly and Met.
 76. The vector of claim 47, wherein theaction domain induces cell death.
 77. The vector of claim 47, whereinthe action domain induces gene transcription.
 78. The vector of claim65, wherein the intracellular localizing domain comprises a secretoryleader sequence, a membrane retention domain, a nuclear localizationdomain, or a vesicle targeting domain.
 79. The vector of any of claims47, 51, 70, 53, 54, 55, 56, 57, 58, 73, 59, 62, 64 or 68 wherein thegenetic construct encodes a chimeric protein comprising as anintracellular domain at least one ligand-binding domain.
 80. The vectorof any of claims 47, 51, 70, 53, 54, 55, 56, 57, 58, 73, 59, 62, 64 or68 wherein the ligand-binding domain is a ligand-binding domain of anintracellular protein.
 81. The vector of any of claims 47, 51, 70, 53,54, 55, 56, 57, 58, 73, 59, 62, 64 or 68, wherein the action domain ofthe chimeric protein comprises:(a) a DNA binding domain; (b) atranscriptional activation domain; (c) a transcriptional repressordomain; or (d) a signaling domain which induces a detectable biologicalprocess following oligomerization of the chimeric protein molecule toform the ligand cross-linked protein complex.
 82. The vector of claim 75wherein the chimeric protein comprises at least one FKBP12 variant inwhich Phe36 or Asp37 or both are replaced with a different amino acidindependently selected from Val or Ala.
 83. The vector of claim 75wherein the chimeric protein comprises at least one FKBP12 variant inwhich Phe36 is replaced with valine.
 84. The vector of claim 78, whereinthe membrane retention domain comprises a membrane spanning domain orlipid membrane binding domain.
 85. A cell comprising at least onegenetic construct encoding a chimeric protein comprising (a) at leastone ligand-binding domain which binds to a selected ligand so as tooligomerize two or more chimeric protein molecules to form a ligandcross-linked complex, the selected ligand having one or more of thefollowing characteristics:(i) the ligand is not a protein; (ii) theligand has a molecular weight less than 5 kD; and (iii) the ligand iscell permeable, and(b) an action domain which is heterologous withrespect to the ligand-binding domain, and which induces a biologicalprocess as a result of the formation of the ligand cross-linked proteincomplex.
 86. The cell of claim 85 which further comprises a target geneunder the expression control of a transcriptional control elementresponsive to formation of said ligand cross-linked complex, and whichfollowing exposure to the selected ligand expresses the target gene. 87.The cell of claim 85 the first chimeric protein includes a DNA-bindingdomain, and which cell further includes (a) a second genetic constructencoding a second chimeric protein including a transcriptionalactivating domain and at least one ligand-binding domain which binds tothe ligand to form a ligand cross-linked complex including both chimericproteins, and (b) a target gene under the transcriptional control of aheterologous transcriptional control sequence which binds with theDNA-binding domain and is responsive to the transcriptional activatingdomains which cell expresses the target gene following exposure to asubstance containing the ligand.
 88. The cell of claim 85, wherein thegenetic construct encodes a chimeric protein comprising two or moreligand-binding domains.
 89. The cell of claim 85, wherein the geneticconstruct encodes a chimeric protein comprising at least oneligand-binding domain which binds to a ligand having a molecular weightless than 3 kD.
 90. The cell of claim 85, wherein the genetic constructencodes a chimeric protein comprising at least one ligand-binding domainhaving between so and 350 amino acid residues.
 91. The cell of claim 85wherein the genetic construct encodes a chimeric protein comprising atleast one naturally-occurring ligand binding domain.
 92. The cell ofclaim 85 wherein the genetic construct encodes a chimeric proteincomprising at least one ligand-binding domain comprising anon-naturally-occurring peptide sequence.
 93. The cell of claim 85wherein the genetic construct encodes a chimeric protein which binds tothe selected ligand with a kD value less than or equal to about 10⁻⁶ M.94. The cell of claim 85 wherein the genetic construct encodes achimeric protein comprising at least one ligand binding domaincomprising an antibiotic binding domain, or antibody domain.
 95. Thecell of claim 85, wherein said ligand comprises a macrocyclic compound.96. The cell of claim 85 wherein the genetic construct encodes achimeric protein comprising at least one ligand-binding domain whichbinds to FK506, FK520, rapamycin, or derivatives thereof.
 97. The cellof claim 85, wherein said ligand is a synthetic organic molecule havinga molecular weight of less than 5 kDa.
 98. The cell of claim 85, whereinsaid ligand is membrane permeable.
 99. The cell of claim 85, wherein theaction domain induces a biological process selected from the groupconsisting of channel opening, ion release, acylation, methylation,hydrolysis, phosphorylation, dephosphorylation, change in redox states,and rearrangement reactions.
 100. The cell of claim 85, wherein theaction domain includes a cytoplasmic domain, or a portion thereof whichinduces a detectable biological process, of a transmembrane receptorprotein.
 101. The cell of claim 85, wherein the action domain has anenzymatic activity selected from the group consisting of a phosphataseactivity, a reductase activity, a cyclooxygenase activity and a proteaseactivity.
 102. The cell of claim 85, wherein the genetic constructencodes a chimeric protein comprising an intracellular localizing domainwhich directs the chimeric protein to a given cellular location. 103.The cell of claim 85, wherein the action domain includes a cytoplasmicdomain, or a subunit or portion thereof which induces a detectablebiological response, of a receptor selected from the group consisting ofCD3z, CD3η, CD3g, CD3d, CD3e, an interferon receptor, an interleukinreceptor, a GM-CSF receptor, a LIF receptor, a CNTF receptor, anoncostatin M receptor, a TGF-β receptor, an EGF receptor, ATR2/neu, aHER2/neu, a HER3/c-erbB-3, Xmrk, an insulin receptor, an IGF-1 receptor,IRR, PDGF receptor, a CSF-1 receptor, c-kit, STK-1/flk-2, an FGFreceptor, flg, bek, an NGF receptor, Ig-alpha/MB-1, Ig-beta/B29, Ror1and Ror2.
 104. The cell of claim 85, wherein the action domain isderived from a cytoplasmic domain of a cell surface receptor selectedfrom the group consisting of a tyrosine kinase receptor, a cytokinereceptor and a growth factor receptor.
 105. The cell of claim 85,wherein the action domain of the chimeric protein induces, uponoligomerization, gene transcription from a gene comprising a DNA elementselected from the group consisting of a cAMP responsive element, an SRE,a VL30, an RSRF, an ISRE, a GAS, an ARRE-1 and an ARRE-2.
 106. A cell ofclaim 87, wherein at least one of the ligand-binding domains of thesecond chimeric protein is different from the ligand-binding domain(s)of the first chimeric protein.
 107. The cell of claim 88, wherein thegenetic construct encodes a chimeric protein comprising two or moredifferent ligand-binding domains.
 108. The cell of claim 90, wherein thegenetic construct encodes a chimeric protein comprising at least oneligand binding domain having less than 200 amino acid residues.
 109. Thecell of claim 93 wherein the generic construct encodes a chimericprotein which binds to the selected ligand with a kD value less than orequal to about 10⁻⁸ M.
 110. The cell of claim 95, wherein themacrocyclic compound is a macrolide.
 111. The cell of claim 96, whereinthe chimeric protein comprises at least one ligand-binding domaincomprising an FK506 binding protein ("FKBP") 12 or a variant thereof inwhich one or more of Tyr26, Phe36, Asp37, Try82, and Phe99 are replacedwith a different amino acid.
 112. The cell of claim 96, wherein thechimeric protein comprises at least one ligand-binding domain comprisingan FKBP12 or a variant thereof in which one or more of Tyr26, Phe36,Asp37, Try82, and Phe99 are replaced with different amino acidsindependently selected from Val, Ala, Gly and Met.
 113. The cell ofclaim 85, wherein the action domain induces cell death.
 114. The cell ofclaim 85, wherein the action domain induces gene transcription.
 115. Thecell of claim 102, wherein the intracellular localizing domain comprisesa secretory leader sequence, a membrane retention domain, a nuclearlocalization domain, or a vesicle targeting domain.
 116. The cell of anyof claims 39, 45, 46, 24, 26, 36, 85, 86, 87 or 106, which cell is amammalian cell.
 117. The cell of any of claims 85, 88, 107, 90, 91, 92,93, 94, 95, 110, 99, 101 or 105, wherein the genetic construct encodes achimeric protein comprising as an intracellular domain at least oneligand-binding domain.
 118. The cell of any of claims 85, 88, 107, 90,91, 92, 93, 94, 95, 110, 99, 101 or 105, wherein the ligand-bindingdomain is a ligand-binding domain of an intracellular protein.
 119. Thecell of any of claims 85, 88, 107, 90, 91, 92, 93, 94, 95, 110, 99, 101or 105, wherein the action domain of the chimeric protein comprises:(a)a DNA binding domain; (b) a transcriptional activation domain; (c) atranscriptional repressor domain; or (d) a signaling domain whichinduces a detectable biological process following oligomerization of thechimeric protein molecule to form the ligand cross-linked proteincomplex.
 120. The cell of claim 112, wherein the chimeric proteincomprises at least one FKBP12 variant in which Phe36 or Asp37 or bothare replaced with a different amino acid independently selected from Valor Ala.
 121. The cell of claim 112, wherein the chimeric proteincomprises at least one FKBP12 variant in which Phe36 is replaced withvaline.
 122. The cell of claim 115, wherein the membrane retentiondomain comprises a membrane spanning domain or lipid membrane bindingdomain.
 123. The cell of claim 116, which cell is a human cell.
 124. Thecell of claim 116 or 123, wherein the cell is a cell type selected fromthe group consisting of neural, mesenchymal, cutaneous, mucosal,stromal, spleen, reticuloendothelial, epithelial, endothelial, hepatic,kidney, gastrointestinal and pulmonary cells.
 125. The cell of claim116, wherein the cell is a hematopoietic cell type of lymphoid ormyelomonocytic lineage.
 126. The cell of claim 116, wherein the cell isa stem cell or a progenitor cell.
 127. The cell of claim 125, whereinthe cell is a hematopoietic cell type selected from the group consistingof T cells, B cells, macrophages and monocytes.
 128. A compositioncomprising two genetic constructs which encode first and second chimericproteins, each chimeric protein individually comprising (a) at least oneligand-binding domain which binds to a selected ligand so as tooligomerize the first and second chimeric protein molecules to form aligand cross-linked protein complex, the selected ligand having one ormore of the following characteristics:(i) the ligand is not a protein;(ii) the ligand has a molecular weight less than 5 kD; and (iii) theligand is membrane permeable, and(b) an action domain which isheterologous with respect to the ligand-binding domain, and whichinduces a biological process as a result of the formation of the ligandcross-linked protein complex.
 129. The composition of claim 128, whereinat least one of the chimeric proteins includes a ligand binding domaincomprising an antibiotic binding domain, or antibody domain.
 130. Thecomposition of claim 128, wherein said ligand comprises a macrocycliccompound.
 131. The composition of claim 128, wherein the action domainof at least one of the chimeric proteins induces a biological processselected from the group consisting of channel opening, ion release,acylation, methylation, hydrolysis, phosphorylation, dephosphorylation,change in redox states, and rearrangement reactions.
 132. Thecomposition of claim 128, wherein at least one of the chimeric proteinincludes an intracellular localizing domain which directs the chimericprotein to a given cellular location.
 133. The composition of claim 128,wherein the action domain of at least one of the chimeric proteins isderived from a cytoplasmic domain of a cell surface receptor selectedfrom the group consisting of a tyrosine kinase receptor, a cytokinereceptor and a growth factor receptor.
 134. The composition of claim128, wherein the first chimeric protein includes a DNA-binding domainand the second chimeric protein includes a transcriptional activationdomain, wherein the ligand cross-linked complex including both chimericproteins controls transcription of a gene having a transcriptionalcontrol sequence which binds with the DNA-binding domain.
 135. Thecomposition of claim 130, wherein the macrocyclic compound is amacrolide.
 136. The composition of claim 128, wherein the action domaininduces cell death.
 137. The composition of claim 128, wherein theaction domain induces gene transcription.
 138. The composition of claim132, wherein the intracellular localizing domain comprises a secretoryleader sequence, a membrane retention domain, a nuclear localizationdomain, or a vesicle targeting domain.
 139. The composition of claim138, wherein the membrane retention domain comprises a membrane spanningdomain or lipid membrane binding domain.
 140. The composition of any ofclaims 128, 129, 130 or 135, wherein the chimeric proteins each include,as an intracellular domain, at least one ligand-binding domain.
 141. Thecomposition of any of claims 128, 129, 130 or 135, wherein theligand-binding domain is a ligand-binding domain of an intracellularprotein.
 142. The composition of any of claims 128, 129, 130 or 135,wherein the action domain of at least one of the chimeric proteincomprises:(a) a DNA binding domain; (b) a transcriptional activationdomain; (c) a transcriptional repressor domain; or (d) a signalingdomain which induces a detectable biological process followingoligomerization of the chimeric protein molecules to form the ligandcross-linked protein complex.
 143. The genetic construct of claim 1which encodes a chimeric protein comprising at least one ligand-bindingdomain comprising an immunophilin domain.
 144. The genetic construct ofclaim 1 which encodes a chimeric protein comprising at least oneligand-binding domain comprising a cyclophilin domain.
 145. The geneticconstruct of claim 1 which encodes a chimeric protein comprising atleast one ligand-binding domain comprising a steroid binding domain.146. The genetic construct of claim 1 which encodes a chimeric proteincomprising at least one ligand-binding domain which binds to rapamycinor derivatives thereof.
 147. The vector of claim 50, wherein the viralvector is an adeno-associated viral vector.
 148. The vector of claim 50,wherein the viral vector is a Herpes simplex viral vector.
 149. Thevector of claim 50, wherein the viral vector is a retroviral vector.150. The vector of claim 47 wherein the genetic construct encodes achimeric protein comprising at least one ligand binding domaincomprising an immunophilin domain.
 151. The vector of claim 47 whereinthe genetic construct encodes a chimeric protein comprising at least oneligand binding domain comprising a cyclophilin domain.
 152. The vectorof claim 47 wherein the genetic construct encodes a chimeric proteincomprising at least one ligand binding domain comprising a steroidbinding domain.
 153. The vector of claim 47, wherein the action domainhas a kinase activity.
 154. The cell of claims 116 or 123, wherein thecell is a hematopoietic cell.
 155. The cell of claims 116 or 123,wherein the cell is a muscle cell.
 156. The cell of claim 85 wherein thegenetic construct encodes a chimeric protein comprising at least oneligand binding domain comprising an immunophilin domain.
 157. The cellof claim 85 wherein the genetic construct encodes a chimeric proteincomprising at least one ligand binding domain comprising a cyclophilindomain.
 158. The cell of claim 85 wherein the genetic construct encodesa chimeric protein comprising at least one ligand binding domaincomprising a steroid binding domain.
 159. The cell of claim 85, whereinthe action domain has a kinase activity.
 160. The composition of claim128, wherein at least one of the chimeric proteins includes a ligandbinding domain comprising an immunophilin domain.
 161. The compositionof claim 128, wherein at least one of the chimeric proteins includes aligand binding domain comprising a cyclophilin domain.
 162. Thecomposition of claim 128, wherein at least one of the chimeric proteinsincludes a ligand binding domain comprising a steroid binding domain.163. The composition of claim 39, wherein one of the chimeric proteinscomprises a transcriptional activation domain, the other chimericprotein comprises a DNA-binding domain, and the target gene is linked toa DNA sequence to which the DNA-binding domain binds.
 164. Thecomposition of claim 39, wherein the target gene encodes a surfacemembrane protein, a secreted protein, a cytoplasmic protein, anantisense message or a ribozyme.
 165. The composition of claim 39,wherein the target gene encodes a hormone, growth factor, interleukin,enzyme or surface membrane protein.